Recombinant collagen and its use in cartilage repair matrices

Abstract

The present invention discloses recombinant collagen and its use in cartilage repair matrices. The recombinant collagen provided by the present invention comprises the sequence shown in SEQ ID No. 1; the amino acid sequence of the recombinant collagen comprises N basic repeating units, each of which contains n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3"; the 3' and 5' ends of the basic repeating units are linked to form the characteristic amino acid sequence. The recombinant collagen provided by the present invention has significant integrin binding activity, and is effective in promoting cell adhesion, proliferation, and differentiation, and repairing cartilage tissue defects, making it suitable for a wide range of applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from (1) an application filed with the China Patent Office on October 8, 2022, application number 2022112245930, and (2) an application filed with the China Patent Office on November 4, 2022, application number 2022113785160. The entire contents of the prior application are incorporated herein by reference.

[0002] The present invention relates to the field of biomaterials, and in particular to recombinant collagen and its use in cartilage repair matrices. [Background technology]

[0003] Articular cartilage injury is a common condition in orthopedics. Trauma, arthritis, and exercise-related injuries can cause articular cartilage damage or loss. Articular cartilage repair is primarily achieved through the proliferation and differentiation of chondrocytes, which produce sufficient extracellular matrix to repair cartilage defects. However, healthy adult chondrocytes are generally quiescent, highly differentiated cells with low vascularization and obtain nutrients primarily from synovial fluid and subchondral bone. Therefore, articular cartilage repair and regeneration after traumatic or pathological damage is very limited. Currently, clinically used treatments include microfracture, osteochondral transplantation, and chondrocyte transplantation. While these methods can repair cartilage defects to some extent and alleviate patient pain, their long-term effects are unsatisfactory. The key reason for this is that the cartilage reconstructed using these methods differs significantly in histology and structure from normal hyaline cartilage, resulting in differences in histological and biomechanical performance compared to normal cartilage.

[0004] In recent years, tissue engineering has made considerable progress in obtaining functional articular cartilage, and several bioengineered therapeutic strategies, including stem cell methods and scaffolding technologies, have been proposed to repair articular cartilage in a minimally invasive and more efficient manner. As one of the three major components of tissue engineering, scaffold materials play a crucial role in the construction of tissue-engineered cartilage, and their mechanical properties and chemical composition influence cell adhesion, proliferation, and cell phenotype. Collagen, with its excellent biocompatibility, bioactivity, and biodegradability, has become a relatively advanced biomaterial for tissue engineering research. However, most conventional collagens are derived from animals. The amino acid sequences of animal-derived collagens are highly similar to those of human collagen (e.g., 95% similarity between porcine and bovine collagens and humans, and 65% similarity between fish and humans). However, these differences can lead to significant immune rejection, sensitization reactions, and the risk of viral infection and transmission when using animal-derived collagen in biomaterials or medical devices. Furthermore, because different subtypes of natural collagen are ubiquitous in tissues and organs and coexist in complex forms, the efficiency of collagen extraction from biological tissues is highly dependent on their natural abundance, and the mixing of different subtypes is unavoidable. Therefore, the deficiencies of animal-derived collagen in terms of batch stability, purity, molecular weight, and distribution further limit their many applications in the biomedical field.

[0005] To solve the above problems, research into the production of recombinant collagen using genetic engineering techniques has been gaining attention. Recombinant collagen is a recombinant protein material that is produced on a large scale using biological fermentation technology after selecting codons with specific functional expression based on the sequence of the natural human collagen gene, appropriately modifying and editing it, and then transcribing it into host cells. Summary of the Invention [Problem to be solved by the invention]

[0006] The problem to be solved by the present invention is to provide a recombinant collagen that has more pronounced integrin binding activity and is effective in promoting cell adhesion, proliferation, and differentiation and repairing cartilage tissue defects, in response to the shortage of animal-derived collagen, and to provide use of the recombinant collagen in a cartilage repair matrix.

[0007] Integrin binding activity is related to the integrin family, a widely distributed family of transmembrane glycoproteins that connect the extracellular matrix to intracellular signaling. Integrins are heterodimers formed from α and β subunits via non-covalent bonds, and currently, at least 24 integrin dimers have been identified, consisting of 18 α and 8 β subunits. The integrin family of cell adhesion molecules (CAMs) is the primary junction between the extracellular matrix and parenchymal cells, such as inflammatory cells and fibroblasts. It is closely related to cell adhesion, spreading, migration, and functional expression, as well as the development, maintenance, and progression of tissue fibrosis, and can regulate interactions such as recognition and adhesion between cells and between cells and the extracellular matrix. [Means for solving the problem]

[0008] The first object of the present invention is to provide a peptide having an amino acid sequence containing N basic repeating units, each of which contains n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3," and wherein the 3' end and 5' end of the basic repeating unit are linked to form the characteristic amino acid sequence.

[0009] The object of the present invention is to provide a recombinant collagen in which N is an integer of 4 or more; and n1 is an integer of 3 or more.

[0010] Furthermore, the N value is an integer of 4 to 300 or more, and further an integer of 4 to 200 or more.

[0011] The N value is set so that the molecular weight of the recombinant collagen is 1 kDa to 250 kDa, and further 3 kDa to 150 kDa.

[0012] Furthermore, the characteristic amino acid sequences may be arranged contiguously or at intervals in the basic repeating unit.

[0013] Furthermore, the amino acid sequence of the recombinant collagen has the following characteristics: [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3) n2 -Yaa2-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa3-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa4-(G-Xaa1-Xaa2-GE-Xaa3) n4 -…………-Yaa n -(G-Xaa1-Xaa2-GE-Xaa3) n -Yaa n+1 -G-Xaa1-Xaa2-] N indicates, wherein Xaa1 is a non-polar hydrophobic amino acid; Xaa2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); and Xaa3 is a basic amino acid.

[0014] Yaa1, Yaa2, Yaa3, Yaa4, ......, Yaa n , Yaa n+1 are each independently selected from none, one or more different or identical amino acids.

[0015] n2, n3, n4, .... n is an integer greater than or equal to 0, and both cannot be 0 at the same time.

[0016] Non-polar hydrophobic amino acids in the present invention include one of glycine (G), valine (V), phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O); and further include one of phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O).

[0017] The basic amino acid includes one of lysine, arginine, and histidine, and is preferably lysine or arginine.

[0018] Furthermore, the recombinant collagen sequence does not contain a protein tag.

[0019] In one embodiment of the present invention, the amino acid sequence of the recombinant collagen is shown in SEQ ID NO.1.

[0020] The second object of the present invention is to obtain recombinant collagen by expressing, extracting, and purifying recombinant collagen in host cells using genetic engineering techniques; the amino acid sequence of the recombinant collagen is linked in such a way that multiple basic repeating units are repeated.

[0021] Furthermore, the amino acid sequence of the recombinant collagen contains N basic repeating units, and the basic repeating units contain n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3"; the 3' end and 5' end of the basic repeating units are linked to form the characteristic amino acid sequence.

[0022] Here, N is an integer of 4 or more; and n1 is an integer of 3 or more.

[0023] A further object of the present invention is to provide a method for preparing the recombinant collagen in which the characteristic amino acid sequences are arranged consecutively or at intervals in the basic repeating unit.

[0024] The N value is an integer from 4 to 300, and further an integer from 4 to 200; including, but not limited to, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30......200.

[0025] Furthermore, the N value is set so that the molecular weight of the recombinant collagen is 1 kDa to 200 kDa, and further 3 kDa to 150 kDa.

[0026] The value of N may be taken from either end of the range defined by the present invention, or from any integer within the range, for example, N may be 1, 3, 10, 50, 70, 90, 95, 100, 125, 150, etc.

[0027] Furthermore, the recombinant collagen sequence does not contain a protein tag.

[0028] The amino acid sequence of the recombinant collagen has the following characteristics: [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3) n2 -Yaa2-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa3-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa4-(G-Xaa1-Xaa2-GE-Xaa3) n4 -…………-Yaa n -(G-Xaa1-Xaa2-GE-Xaa3) n -Yaa n+1 -G-Xaa1-Xaa2-] N indicates, wherein Xaa1 is a non-polar hydrophobic amino acid; Xaa2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); and Xaa3 is a basic amino acid.

[0029] Yaa1, Yaa2, Yaa3, Yaa4, ......, Yaa n , Yaa n+1 are each independently selected from none, one or more different or identical amino acids.

[0030] n2, n3, n4, .... n is an integer greater than or equal to 0, and both cannot be 0 at the same time.

[0031] A third object of the present invention is to provide a use of said recombinant collagen in the preparation of a product for controlling cell adhesion, proliferation and differentiation.

[0032] A fourth object of the present invention is to provide the use of said recombinant collagen in the preparation of a cartilage repair matrix product.

[0033] When applied to the above-mentioned uses, the recombinant collagen of the present invention may be used in combination with a polymer and / or its derivative, and the polymer includes natural polymers and synthetic polymers; further, the natural polymers include, but are not limited to, hyaluronic acid, chitosan, alginic acid, cellulose, etc.; and further, the synthetic polymers include, but are not limited to, PLA, PGA, PLCL, and PVA.

[0034] When the recombinant collagen of the present invention is applied to the above uses, the concentration used may be 0.01 to 100 mg / mL, for example, 0.01 mg / mL, 0.05 mg / mL, 2 mg / mL, 4.5 mg / mL, 7 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, etc., and the concentration used depends on the application site and situation.

[0035] A fifth object of the present invention is to provide a cartilage repair matrix comprising a polymer and / or a derivative thereof and the recombinant collagen described above.

[0036] Furthermore, the polymer includes a natural polymer and a synthetic polymer; further, the natural polymer is at least one selected from hyaluronic acid, chitosan, alginic acid, and cellulose; and further, the synthetic polymer is at least one selected from PLA, PGA, PLCL, and PVA.

[0037] A sixth object of the present invention is to provide a method for preparing said cartilage repair matrix, comprising the steps of: (1) The hyaluronic acid derivative is dissolved in a buffer solution, and the EDC / sNHS solution is added to react. (2) The recombinant collagen is added to the reaction system in step (1) and reacted, and the solution obtained after the reaction is dialyzed and dried to obtain a composite matrix. (3) After the composite matrix is ​​dissolved, a photoinitiator solution is added to obtain a cartilage repair matrix.

[0038] Humanized collagen has poor mechanical properties due to its small molecular weight, and is difficult to gel even after denaturation, making it unable to meet the requirements of cartilage tissue engineering. In the present invention, hyaluronic acid is first chemically modified with methacrylic anhydride to introduce unsaturated carbon-carbon double bonds, and then EDC / sNHS is added to activate the carboxyl groups in hyaluronic acid. Recombinant humanized collagen is then added, and the amino groups in the protein and the activated carboxyl groups in hyaluronic acid are coupled through an amidation reaction. Finally, photocrosslinking is performed to prepare a composite hydrogel that not only has good mechanical properties and biocompatibility, but is also easy to operate due to the photocrosslinking method, which can meet the requirements of cartilage tissue engineering and can also meet the clinical requirements of filling irregular defects.

[0039] In the present invention, EDC means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and NHS means N-hydroxysuccinimide.

[0040] Furthermore, the polymer derivative described in step (1) is hyaluronic acid modified with methacrylic anhydride.

[0041] Furthermore, the molecular weight of the denatured hyaluronic acid is 100 to 1500 kDa, preferably 300 to 600 kDa, and more preferably 300 kDa.

[0042] Furthermore, the degree of hyaluronic acid modification and grafting described in step (1) is 20 to 60%, preferably 30 to 50%.

[0043] Furthermore, in step (1), after adding EDC and sNHS, the concentration of sNHS is 0.5 to 5 mM, and the concentration of EDC is 0.5 to 5 mM; further, the concentration of sNHS is 1 to 3 mM, and the concentration of EDC is 2 to 4 mM.

[0044] In the present invention, in step (1), the pH value of the mixed solution after adding the EDC / sNHS solution is 4 to 5, preferably 4.75.

[0045] The pH of the mixed solution after the reaction is 7 to 8, and the reaction time is 2 to 5 hours; preferably, the pH is 7.4, and the reaction time is 3 hours.

[0046] In the present invention, in step (2), the mass ratio of recombinant collagen:polymer derivative is 0.5-30:2-10; further 0.5-20:6, and further 1-11:6.

[0047] Furthermore, the reaction time in step (2) is 5 to 15 hours, preferably 8 to 12 hours.

[0048] Furthermore, in step (2), the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, dialyzed against deionized water for 2 to 4 days, and then freeze-dried to obtain a composite matrix.

[0049] The photoinitiator used in the present invention is at least one of LAP and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and the final concentration is 0.01 to 0.05%, preferably 0.05%.

[0050] In the present invention, the concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg / ml, further 5 to 20 mg / ml, and further 7 to 17 mg / ml.

[0051] The cartilage repair matrix of the present invention is a product containing the recombinant collagen, and can be used as a component of medical devices, including implant materials, tissue engineering scaffold materials, soft tissue filler materials, cell or other active substance carrier materials, etc.

[0052] When the cartilage repair matrix of the present invention is used clinically, it may be prepared into a cartilage repair hydrogel after adding cells, or it may be applied to a cartilage defect site as is without adding cells.

[0053] The seventh object of the present invention is to provide a method for preparing a cartilage repair hydrogel, which comprises combining the cartilage repair matrix prepared by the above method with chondrocytes or bone marrow mesenchymal stem cells, injecting the resulting mixture into the site of cartilage damage, and irradiating the mixture with light to form a cartilage repair hydrogel.

[0054] The wavelength of the light irradiation is appropriately selected depending on the initiator used.

[0055] The cartilage repair hydrogel of the present invention further comprises a physiologically acceptable carrier material.

[0056] Here, carrier materials include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesteryl stearate, etc.), and enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.).

[0057] The composition can also be used in combination with other functional materials, such as components having antibacterial and bacteriostatic properties, anti-aging components, anticoagulant components, antioxidant components, growth factors, etc., as needed.

[0058] The composition can be prepared by conventional pharmaceutical or cosmetic techniques, which involve mixing the recombinant collagen of the present invention as an active ingredient with a carrier and forming it into a desired dosage form by conventional techniques. The composition of the present invention can be formulated into various dosage forms, including oral administration, mucosal administration, injection, inhalation, external application, etc., as needed.

[0059] The products of the present invention include, but are not limited to, medicines, foods, dietary supplements or cosmetics, and daily necessities. In this specification, the term "possible" is used to mean both cases where some processing is being performed and cases where some processing is not being performed.

[0060] In practical use, the recombinant collagen and the composition of the present invention can be administered directly to a patient as a drug or mixed with an appropriate carrier or excipient. The carrier material can include, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesteryl stearate, etc.), and enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. These materials can be used to formulate various dosage forms, including, but not limited to, tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections. The suppositories can be vaginal suppositories, vaginal rings, or creams, lotions, or gels suitable for vaginal application.

[0061] The dosage form may be a conventional formulation, a sustained-release formulation, a controlled-release formulation, or various microparticle delivery systems. A wide variety of carriers known in the art can be used to make the unit dosage form into tablets. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone; Disintegrants include, for example, dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors include, for example, sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption enhancers include, for example, quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants include, for example, talc, silica, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be coated, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-coated tablets, multi-layered tablets, etc. To prepare the unit dosage form as a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, etc. In order to prepare the unit-dosage form as a suppository, a wide variety of carriers known in the art can be used.Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To prepare unit-dose injectable preparations such as solutions, emulsions, lyophilized powder injections, and suspensions, any diluent commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. To prepare isotonic injections, an appropriate amount of sodium chloride, glucose, or glycerin can be added to the injectable preparation, and conventional solubilizers, buffers, pH adjusters, etc. can also be added. If necessary, colorants, preservatives, fragrances, flavorings, sweeteners, or other materials can also be added to the drug formulation.

[0062] The above dosage forms can be used for injection administration, including subcutaneous, intravenous, intramuscular and intraperitoneal injections, intracisternal injection or infusion; transcavitary administration, such as rectal, vaginal and sublingual administration; respiratory administration, such as via the nasal cavity; and mucosal administration.

[0063] The administration route is preferably injection, and the preferred injection route is subcutaneous injection.

[0064] The dosage of the recombinant collagen and the compositions of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, and individual response of the patient or animal, the specific active ingredient used, the route of administration, and the number of doses. The above-mentioned doses can be administered as a single dose or divided into several doses, for example, 2, 3, or 4 doses. For any particular patient, the specific therapeutically effective dose level will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health, sex, and diet; the administration time, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination or simultaneously with the specific active ingredient used; and similar factors known in the medical field.

[0065] For example, it is common in the art to start dosages of the active ingredient at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. [Effects of the Invention]

[0066] (1) The recombinant collagen provided by the present invention has significant integrin binding activity, promotes cell adhesion, proliferation, and differentiation, and has the effect of repairing cartilage tissue defects, and has favorable prospects for application. (2) The present invention uses covalent crosslinking of hyaluronic acid and humanized collagen to form a stable composite hydrogel matrix, which has good mechanical properties and can maintain the morphology of the composite well; it has good biocompatibility, and the photocrosslinking method makes the operation easier, meeting the requirements of cartilage tissue engineering and also meeting the clinical requirements of filling irregular defects. [Brief explanation of the drawings]

[0067] [Figure 1] This is the result of fibroblast adhesion and proliferation. [Figure 2] OD values ​​of fibroblast adhesion. [Figure 3] This is the result of chondrocyte adhesion and proliferation. [Figure 4] OD values ​​of chondrocyte adhesion. [Figure 5] 1 shows the results of an adhesion experiment using different concentrations of Array A and Array B on human gingival fibroblasts. [Figure 6] Morphology of chondrocyte / hydrogel complexes after 1, 7, 14, and 21 days of in vitro culture. [Figure 7] OD values ​​of chondrocyte adhesion after 1, 7, and 14 days of in vitro culture of chondrocyte / hydrogel complexes. [Figure 8] 1 is a graph of CCK-8 in chondrocytes after 1, 7, and 14 days of in vitro culture of chondrocyte / hydrogel composites. [Figure 9] This shows the staining results of the chondrocyte / hydrogel complex. [Figure 10] This shows the results of type II collagen immunohistochemical staining of the chondrocyte / hydrogel complex. [Figure 11] This shows the results of a quantitative test of GAG in the chondrocyte / hydrogel complex. DETAILED DESCRIPTION OF THE INVENTION

[0068] Hereinafter, embodiments of the present invention will be described with reference to the drawings. These embodiments are based on the technical aspects of the present invention, and provide detailed embodiments and specific operating procedures, but the technical scope of the present invention is not limited to the following examples.

[0069] Throughout this specification, unless otherwise specified, the terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of a conflict, the present specification shall prevail.

[0070] Example 1 A carrier containing a nucleotide sequence encoding the recombinant collagen was constructed, a host cell (which may be a prokaryotic or eukaryotic cell) containing the carrier was constructed and screened, the host cell was cultured under appropriate conditions to express the protein, and the expressed recombinant collagen was collected and purified.

[0071] As used herein, the term "carrier" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A carrier is called an expression carrier if it allows for the expression of a protein encoded by the inserted polynucleotide. A carrier can be introduced into a host cell by transformation, transduction, or transfection to express the genetic material elements it carries in the host cell. Carriers are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; Cox plasmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. A carrier may contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A carrier may also contain a replication origin. A carrier may contain a nucleic acid of the present invention for introduction into a cell and expression. A carrier may also contain expression control elements, such as a promoter, terminator, and / or enhancer, operably linked to the nucleic acid.

[0072] Genetic recombination and transcription in this invention refers to synthesizing a nucleotide sequence encoding a recombinant collagen, cloning the nucleotide sequence into an expression vehicle by conventional methods, transforming the expression vehicle into host cells, and constructing and screening to obtain an engineered strain. Transformation methods include, but are not limited to, electroporation and CaCl2 transformation; the expression vehicle may be a conventional carrier such as pET26, pET32, pGEX-6p, pPIC9, or pPIC9K, or it may be a plasmid, phage, virus, or other carrier.

[0073] The term "host cell" as used herein refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral carriers, transformation with plasmid carriers, and the introduction of naked DNA facilitated by electroporation, lipofection, and particle guns. Host cells may be eukaryotic or prokaryotic. Examples include microbial cells such as Enterobacteriaceae cells (e.g., E. coli BL21, DH5α, etc.), yeast (e.g., Pichia pastoris, Brewer's yeast, etc.), or eukaryotic cells such as CHO cells.

[0074] Fermentation culture according to the present invention refers to the induction of fermentation in a sterile fermenter using an engineered strain that has been selected and confirmed to have high protein expression, together with an appropriate fermentation broth, under appropriate conditions of temperature, pressure, and pH.

[0075] The isolation and purification of the protein according to the present invention involves subjecting the cells to treatments such as lysis, homogenization, and separation to obtain a bacterial solution, and then subjecting the resulting solution to conventional protein isolation and purification techniques, including, but not limited to, filtration, centrifugation, salting out, dialysis, liquid chromatography, ion exchange chromatography, and affinity chromatography.

[0076] In an embodiment of the present invention, genetic recombination and transcription are carried out using E. coli as a host cell, specifically: 1. Preparation of Recombinant Collagen by E. coli Fermentation Genetic engineering and transcription: The DNA fragment was codon-optimized and spliced ​​by PCR, and pET-32a was selected to construct an expression carrier, which was then transferred into the E. coli expression strain BL21. After cultivation and selection, an E. coli genetic engineering strain with high protein expression was obtained.

[0077] Fermentation: A single colony of the screened E. coli engineered strain was picked from the LB plate and placed in a 100mL Erlenmeyer flask containing 10mL of LB medium and cultured at 37℃ and 220 rpm for 12-16 hours. The bacterial solution was inoculated into a fermenter containing LB medium at a ratio of 1:100 for amplification. After culturing at 37℃ and 220 rpm for 3 hours until the OD600 reached approximately 0.6, 0.5mM IPTG was added and the resulting culture was induced at 16℃ for 20 hours, after which the bacterial cells were collected by centrifugation.

[0078] Protein isolation and purification: After reselection of the cells in Tris buffer, the cells were completely lysed by high-speed stirring, and the supernatant was collected by centrifugation and cooled to 4°C. The supernatant was filtered sequentially through 1 μm, 0.45 μm, and 0.22 μm filters, and then further purified by affinity chromatography to obtain recombinant collagen.

[0079] 2. Cell culture method 1) Sample preparation: Recombinant collagen is prepared into a solution of a predetermined concentration (e.g., 0.5 mg / ml) using PBS, and animal-derived collagen solution is diluted to the same concentration (e.g., 0.5 mg / ml) using PBS, with PBS serving as a blank control. 2) Plate distribution: The experimental solution was added to a 96-well plate, 100 μL per well, 5 wells per group, and the plate was left standing at 4° C. overnight. 3) Blocking: After plate distribution was completed, the liquid was removed from the well plate, which was then washed twice with 200 μL of PBS solution. 100 μL of heat-inactivated 1% BSA-PBS solution was added, and the plate was incubated at 37°C in a 5% CO2 incubator for 1 hour. 4) Cell inoculation: After incubation, remove the liquid from the well plate, wash twice with 200 μL of PBS solution, and inoculate each well with 100 μL of 5 × 10 cells at a density of 5 × 10. 4 -1×10 6 A cell suspension at 1000 cells / ml was added and incubated in an incubator at 37°C and 5% CO2 for 1 hour. 5) Detection: After incubation, the liquid was removed from the wells and washed twice with 200 μL of PBS solution. 150 μL of CCK-8 was added and incubated for 1 hour in an incubator at 37°C and 5% CO2. 100 μL of detection solution was then aspirated and added to a new 96-well plate, and the optical density (OD) was detected at 450 nm.

[0080] <Test Example 1> Using the method of Example 1, recombinant collagens having the following three different amino acid sequences were prepared.

[0081] Recombinant collagen obtained by directly linking 16 repeat units having multiple combinations of the above characteristic amino acid sequences improves a series of experiments such as cell adhesion, migration, and functional expression. Sequence A: GER GAP GFR GPA GPN GIP GEK GPA GER GAP is linked 16 times, and its full sequence is as follows (SEQ ID NO. 1): GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP GERGAPGFRG PAGPNGIPGE KGPAGERGAP.

[0082] Another recombinant collagen obtained by directly repeating four repeating units having the above characteristic amino acid sequence has different cell adhesion and proliferation activities. Sequence B: GEK GSP GAD GPA GAP GTP GPQ GIA GQR GVV GLP GQR GER GFP GLP GPS GEP GKQ GPS GAS is linked four times, and its full sequence is as follows (SEQ ID NO. 2): GEKGSPGADG PAGAPGTPGP QGIAGQRGVV GLPGQRGERG FPGLPGPSGE PGKQGPSGAS GEKGSPGADG PAGAPGTPGP QGIAGQRGVV GLPGQRGERG FPGLPGPSGE PGKQGPSGAS GEKGSPGADG PAGAPGTPGP QGIAGQRGVV GLPGQRGERG FPGLPGPSGE PGKQGPSGAS GEKGSPGADG PAGAPGTPGP QGIAGQRGVV GLPGQRGERG FPGLPGPSGE PGKQGPSGAS.

[0083] Recombinant collagen in which the above repeating unit is linked 16 times exhibits significantly reduced cell adhesion and proliferation activity when a tag is added. Sequence C: HHHHHH GER GAP GFR GPA GPN GIP GEK GPA GER GAP is linked 16 times, and its full sequence is as follows (SEQ ID NO. 3): HHHHHHGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAPGERG APGFRGPAGP NGIPGEKGPA GERGAP.

[0084] The above three types of collagen were subjected to fibroblast adhesion, chondrocyte adhesion tests, and human gingival fibroblast adhesion promotion tests using the cell culture method of Example 1. The experimental results are shown in Figures 1 to 3.

[0085] As can be seen from the results in Figure 1, in the recombinant collagen experimental group obtained by repeatedly linking sequence A, which has a combination of many characteristic amino acid sequences, clear fibroblast adhesion was observed and cell morphology was good; the recombinant collagen experimental group obtained by repeatedly linking sequence B, which has a reduced characteristic amino acid sequence, and the animal collagen group had similar fibroblast adhesion and cell morphology; the recombinant collagen group obtained by repeatedly linking sequence C, which had a tag added, showed a clear decrease in fibroblast adhesion, and the blank control group showed little fibroblast adhesion.

[0086] As can be seen from the results in Figure 2, the experimental group of recombinant collagen obtained by repeatedly linking sequence A, which has a combination of many characteristic amino acid sequences, most strongly promoted fibroblast adhesion, followed by the recombinant collagen obtained by repeatedly linking animal collagen and sequence B. The adhesion levels of fibroblasts in all three groups were significantly higher than those of the recombinant collagen obtained by repeatedly linking sequence C and the blank control; the adhesion level of fibroblasts in the recombinant collagen group with sequence C was slightly higher than that of the blank control.

[0087] As can be seen from the results in Figure 3, chondrocyte adhesion was most pronounced in the animal collagen group, filling almost the entire field of view; next was the recombinant collagen group with sequence A, where chondrocyte adhesion was evident and relatively concentrated, with good cell morphology; in the recombinant collagen group with sequence B, the adhered chondrocytes were relatively dispersed, with only some concentrated, and with good cell morphology; in the recombinant collagen group with sequence C, the adhered chondrocytes were dispersed and few in number; and in the control blank, only a small number of chondrocytes adhered and were dispersedly distributed.

[0088] As can be seen from the results in Figure 4, animal collagen contributed most to promoting chondrocyte adhesion, significantly higher than the other groups; the recombinant collagen group of sequence A, which has many characteristic amino acid sequence combinations, was significantly higher than the recombinant collagen group of sequence B, the recombinant collagen group of sequence C, and the blank control group; the recombinant collagen group of sequence B was significantly higher than the recombinant collagen group of sequence C and the blank control group; and the recombinant collagen group of sequence C was slightly higher than the control group, but there was no statistical difference.

[0089] As can be seen from the results of the human gingival fibroblast adhesion promotion experiment in Figure 5, the recombinant collagen group of sequence A, which has many characteristic amino acid sequence combinations, is more advantageous for human gingival fibroblast adhesion than sequence B at all concentrations; as the concentration of recombinant collagen increases, the adhesion of human gingival fibroblasts also gradually increases.

[0090] Example 2: Use of recombinant collagen in promoting cartilage repair Using the method of Example 1, a recombinant collagen conforming to the amino acid sequence characteristics of the present invention was prepared. The core amino acid sequence of this recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeated 16 times and used to promote cartilage repair. The specific steps are: (1) Methacrylated hyaluronic acid (HA-MA) with a molecular weight of 300 kDa and a degree of denaturation of 37% was dissolved in MES buffer to obtain a 1 mM solution of methacrylated hyaluronic acid. (2) Solid sNHS and solid EDC were added sequentially to the mixed solution so that the sNHS concentration was 2 mM and the EDC concentration was 3 mM, and the pH of the mixed solution was maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution. (3) The reaction was carried out at room temperature for 2 hours with stirring. (4) The pH of the reaction system was adjusted to 7.4 with 1.0 M NaOH solution. (5) Recombinant humanized collagen was added to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution was 1:6, and the reaction was carried out with stirring for 8 hours. (6) The solution after the reaction was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, and dialyzed against deionized water for 3 days. The bag was then freeze-dried in a freeze-dryer to obtain freeze-dried sponge HA-rhCol III. (7) The lyophilized sponge from step (6) was dissolved in 0.01 M PBS, and LAP photoinitiator solution (final concentration 0.05% by mass / volume) was added so that the final concentration of the HA-rhCol III solution was 7 mg / ml. (8) The precursor solution from step (7) was poured into a silica gel mold with a standard diameter of 6 mm × 2.5 mm and irradiated with ultraviolet light for 1 minute to obtain a recombinant humanized collagen-containing hyaluronic acid hydrogel (HA-rhCol III).

[0091] Example 3: Use of recombinant collagen in promoting cartilage repair Using the method of Example 1, a recombinant collagen conforming to the amino acid sequence characteristics of the present invention was prepared. The core amino acid sequence of this recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeated 16 times and used to promote cartilage repair. The specific steps are: (1) Methacrylated hyaluronic acid (HA-MA) with a molecular weight of 100 kDa and a degree of denaturation of 50% was dissolved in MES buffer to obtain a 2 mM solution of methacrylated hyaluronic acid. (2) Solid sNHS and solid EDC were added sequentially to the mixed solution so that the concentration of sNHS was 3 mM and the concentration of EDC was 5 mM, and the pH of the mixed solution was maintained at 4.75 with 0.5 M NaOH solution or 0.5 M HCl solution. (3) The reaction was carried out at room temperature for 2 hours with stirring. (4) The pH of the reaction system was adjusted to 7.4 with 1.0 M NaOH solution. (5) Recombinant humanized collagen was added to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution was 11:6, and the reaction was carried out with stirring for 12 hours. (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed against deionized water for 3 days, and then freeze-dried in a freeze dryer to obtain the product HA-rhCol III. (7) The lyophilized sponge from step (6) was dissolved in 0.01 M PBS, and LAP photoinitiator solution (final concentration 0.01% by mass / volume) was added so that the final concentration of the HA-rhCol III solution was 17 mg / ml. (8) The precursor solution from step (7) was poured into a silica gel mold with a dimension of Φ6 mm × 2.5 mm and irradiated with ultraviolet light for 1 min to obtain HA-rhCol III hydrogel.

[0092] Example 4: Use of recombinant collagen in promoting cartilage repair Using the method of Example 1, a recombinant collagen conforming to the amino acid sequence characteristics of the present invention was prepared. The core amino acid sequence of this recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is linked 16 times (SEQ ID NO. 1), and used to promote cartilage repair. The specific steps are: (1) Methacrylated hyaluronic acid (HA-MA) with a molecular weight of 600 kDa and a degree of denaturation of 30% was dissolved in MES buffer to obtain a 1 mM solution of methacrylated hyaluronic acid. (2) sNHS and EDC were added to the mixed solution in that order so that the sNHS concentration was 1 mM and the EDC concentration was 3 mM, and the pH of the mixed solution was maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution. (3) The reaction was carried out at room temperature for 3 hours with stirring. (4) The pH of the reaction system was adjusted to 7.4 with 1.0 M NaOH solution. (5) Recombinant humanized collagen was added to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution was 0.5:6, and the reaction was carried out with stirring for 8 hours. (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed against deionized water for 3 days, and then freeze-dried in a freeze dryer to obtain the product HA-rhCol III. (7) The lyophilized sponge from step (6) was dissolved in 0.01 M PBS, and LAP photoinitiator solution (final concentration 0.05% by mass / volume) was added so that the final concentration of the HA-rhCol III solution was 7 mg / ml. (8) The precursor solution from step (7) was poured into a silica gel mold with a dimension of Φ6 mm × 2.5 mm and irradiated with ultraviolet light for 2 min to obtain HA-rhCol III hydrogel.

[0093] Example 5: Use of recombinant collagen in promoting cartilage repair Using the method of Example 1, a recombinant collagen conforming to the amino acid sequence characteristics of the present invention was prepared. The core amino acid sequence of this recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP, which is repeated 16 times (SEQ ID NO. 1), and used to promote cartilage repair. The specific steps are: (1) Methacrylated hyaluronic acid (HA-MA) with a molecular weight of 1000 kDa and a degree of denaturation of 40% was dissolved in MES buffer to obtain a 0.6 mM methacrylated hyaluronic acid solution. (2) sNHS and EDC were added sequentially to the mixed solution so that the concentration of sNHS was 5 mM and the concentration of EDC was 5 mM, and the pH of the mixed solution was maintained at 4.75 with 0.2 M NaOH solution or 0.2 M HCl solution. (3) The reaction was carried out at room temperature for 3 hours with stirring. (4) The pH of the reaction system was adjusted to 7.4 with 0.5 M NaOH solution. (5) Recombinant humanized collagen was added to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution was 11:6, and the reaction was carried out with stirring for 12 hours. (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed against deionized water for 3 days, and then freeze-dried in a freeze dryer to obtain the product HA-rhCol III. (7) The lyophilized sponge from step (6) was dissolved in 0.01 M PBS, and I2959 photoinitiator solution (final concentration 0.03% by mass / volume) was added to give a final concentration of 17 mg / ml of HA-rhCol III solution. (8) The precursor solution from step (7) was poured into a silica gel mold with a dimension of Φ6 mm × 2.5 mm and irradiated with ultraviolet light for 1 min to obtain HA-rhCol III hydrogel.

[0094] <Test Example 2> The recombinant humanized collagen-modified hyaluronic acid hydrogel prepared in Example 2 gave the following results in cell experiments.

[0095] In conjunction with the results in Figures 6 and 7, when chondrocytes were cultured in HA-MA hydrogels for up to 21 days, the lack of adhesion sites led to in situ aggregation and growth of chondrocytes. Furthermore, in the HA-rhCol III hydrogel, chondrocytes became partially dispersed after 7 days of culture, but the diffusion of cells became more evident at 14 and 21 days, with the distribution becoming more uniform. These results suggest that rhCol III is favorable for cell adhesion, migration, and proliferation, and that HA-rhCol III hydrogels promote chondrocyte proliferation more than HA-MA hydrogels.

[0096] The results in Figure 8 show that the HA-rhCol III hydrogels showed no deformation or condensation of cell nuclei, and the actin of the cells in both hydrogels was spindle-shaped after 7 and 14 days of culture, indicating good cell spreading within the gel. In the HA-MA group, the cells proliferated in situ in the form of cell clusters at each time point. These results indicate that rhCol III provides suitable sites for cell adhesion, allowing for faster adhesion of chondrocytes and providing the conditions for cell proliferation.

[0097] Histological and type II collagen immunohistochemistry results (Figures 9-10) showed that the HA-rhCol III gel had more prominent polysaccharide and protein staining regions, indicating that the addition of rhCol III may promote functional chondrocyte expression. Quantitative analysis of GAG (Figure 11) confirmed that the addition of rhCol III further promoted the secretion of extracellular matrix cartilage.

[0098] The above description of the embodiments is provided to enable those skilled in the art to understand and use the present invention. It will be apparent that various modifications to these embodiments will be readily apparent to those skilled in the art, and that the general principles described herein can be applied to other embodiments without the need for inventive effort. Therefore, the present invention is not limited to the above-described embodiments, and it is apparent that those skilled in the art can modify or substitute the embodiments without departing from the spirit and scope of the present invention.

Claims

1. The amino acid sequence contains N basic repeating units, and the basic repeating units contain the following characteristic amino acid sequence: "G-Xaa １ -Xaa ２ -G-E-Xaa ３ "; the 3'-end and 5'-end of the basic repeating unit are linked to form the characteristic amino acid sequence; wherein N is an integer of 4 or more; and n1 is an integer of 3 or more.

2. The recombinant collagen according to claim 1, wherein N is an integer of 4 to 300.

3. The recombinant collagen according to claim 1, wherein N is an integer of 4 to 200.

4. 2. The recombinant collagen according to claim 1, wherein the characteristic amino acid sequences are arranged contiguously or at intervals in the basic repeating unit.

5. The amino acid sequence of the recombinant collagen has the following characteristics: [!!!!a ３ ________________ １ (!︁] １ ︁︁ ２ _______________________________ ３ ) ｎ２ ________________ ２ (!︁] １ ︁︁ ２ _______________________________ ３ ) ｎ３ ________________ ３ (!︁] １ ︁︁ ２ _______________________________ ３ ) ｎ３ ________________ ４ (!︁] １ ︁︁ ２ _______________________________ ３ ) ｎ４ ............1 ｎ (!︁] １ ︁︁ ２ _______________________________ ３ ) ｎ ________________ ｎ＋１ ___________________ １ ︁ ２  Ｎ をPlease、、 However, Xaa １ is a non-polar hydrophobic amino acid; ２ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); ３ is a basic amino acid; Yaa １ , Yaa ２ , Yaa ３ , Yaa ４ , ………, Yaa ｎ , Yaa ｎ＋１ are each independently selected from none, one or more different or identical amino acids; The recombinant collagen according to claim 1, characterized in that n2, n3, n4, ..., n are integers of 0 or more, and both cannot be 0 at the same time.

6. The recombinant collagen of claim 1, characterized in that the recombinant collagen sequence does not contain a protein tag.

7. The recombinant collagen according to any one of claims 1 to 6, characterized in that the recombinant collagen sequence is shown in SEQ ID NO.

1.

8. The method for preparing recombinant collagen according to any one of claims 1 to 6, characterized in that the recombinant collagen is expressed, extracted, and purified by a host cell using genetic engineering technology; and the amino acid sequence of the recombinant collagen is linked so as to repeat a plurality of basic repeating units.

9. The amino acid sequence of the recombinant collagen contains N basic repeating units, and the basic repeating units contain the following characteristic amino acid sequence: "G-Xaa １ -Xaa ２ -G-E-Xaa ３ "; the 3'-end and 5'-end of the basic repeating unit are linked to form the characteristic amino acid sequence; 9. The method for preparing recombinant collagen according to claim 8, wherein N is an integer of 4 or more; and n1 is an integer of 3 or more.

10. 10. The method for preparing recombinant collagen according to claim 9, wherein the characteristic amino acid sequences are arranged consecutively or at intervals in the basic repeating unit.

11. the recombinant collagen sequence does not contain a protein tag; The amino acid sequence of the recombinant collagen has the following characteristics: [!!!!a ３ ________________ １ (!︁] １ ︁ ２ _______________________________ ３ ) ｎ２ ________________ ２ (!︁] １ ︁ ２ _______________________________ ３ ) ｎ３ ________________ ３ (!︁] １ ︁ ２ _______________________________ ３ ) ｎ３ ________________ ４ (!︁] １ ︁ ２ _______________________________ ３ ) ｎ４ ............1 ｎ (!︁] １ ︁ ２ _______________________________ ３ ) ｎ ________________ ｎ＋１ ___________________ １ ︁ ２  Ｎ をPlease、、 However, Xaa １ is a non-polar hydrophobic amino acid; １ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); ３ is a basic amino acid; Yaa １ , Yaa ２ , Yaa ３ , Yaa ４ , ………, Yaa ｎ , Yaa ｎ＋１ are each independently selected from none, one or more different or identical amino acids; 9. The method for preparing recombinant collagen according to claim 8, wherein n2, n3, n4, ...., n are integers of 0 or more, and both cannot be 0 at the same time.

12. Use of the recombinant collagen according to any one of claims 1 to 7 in the preparation of a product for controlling cell adhesion, proliferation and differentiation.

13. Use of a recombinant collagen according to any one of claims 1 to 7 in the preparation of a cartilage repair matrix product.

14. A cartilage repair matrix comprising a polymer and / or a derivative thereof and the recombinant collagen according to any one of claims 1 to 7; the polymer includes a natural polymer and a synthetic polymer.

15. The cartilage repair matrix of claim 14, wherein the natural polymer is at least one selected from the group consisting of hyaluronic acid, chitosan, alginic acid, and cellulose; and the synthetic polymer is at least one selected from the group consisting of PLA, PGA, PLCL, and PVA.

16. (1) dissolving a polymer derivative in a buffer solution, and adding an EDC / sNHS solution to react with the polymer derivative; (2) adding the recombinant collagen according to any one of claims 1 to 7 to the reaction system in step (1) to react, and then dialyzing and drying the solution obtained after the reaction to obtain a composite matrix; (3) After dissolving the composite matrix, adding a photoinitiator solution to obtain a cartilage repair matrix; and 16. A method for preparing a cartilage repair matrix according to claim 15, comprising:

17. The polymer derivative described in step (1) is hyaluronic acid modified with methacrylic anhydride; the molecular weight of the modified hyaluronic acid is 100-1500 kDa; the degree of modification and grafting of the hyaluronic acid is 20-60%; the pH of the mixed solution after adding the EDC / sNHS solution is 4-5; after adding EDC and sNHS, the concentration of sNHS is 0.5-5 mM and the concentration of EDC is 0.5-5 mM; the pH of the mixed solution after the reaction is 7-8, and the reaction time is 2-5 hours; In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10; the reaction time is 5-15 hours; the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8,000-14,000, dialyzed with deionized water for 2-4 days, and then freeze-dried to obtain a composite matrix; The method for preparing a cartilage repair matrix according to claim 16, wherein the concentration of the composite matrix after adding the photoinitiator in step (3) is 2-30 mg / ml.

18. In step (1), the molecular weight of the hyaluronic acid after modification is 300-600 kDa; the degree of hyaluronic acid modification and grafting is 30-50%; In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-20:6, and the reaction time is 8-12 hours; 18. The method for preparing a cartilage repair matrix according to claim 17, wherein in step (3), the concentration of the composite matrix after adding the photoinitiator is 5-20 mg / ml.

19. A method for preparing a cartilage repair hydrogel, comprising: combining a cartilage repair matrix prepared by the method according to any one of claims 16 to 18 with chondrocytes or bone marrow mesenchymal stem cells; injecting the resulting mixture into a site of cartilage damage; and irradiating the resulting mixture with light to form a cartilage repair hydrogel.

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