A micro-cross-linked recombinant collagen articular cartilage repair gel and its preparation method

Abstract

The present invention relates to a micro-crosslinked recombinant collagen articular cartilage repair gel and a preparation method thereof, wherein the gel is obtained by chemically crosslinking recombinant type II humanized collagen, sodium hyaluronate and chondroitin sulfate through EDC / NHS; wherein, by mass, the raw materials include: 8-20 parts of recombinant type II humanized collagen, 0.5-2 parts of sodium hyaluronate, and 0.3-1 parts of chondroitin sulfate. The gel has the characteristics of non-immunogenicity, non-toxicity, non-pyrogenicity and high activity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a micro-cross-linked recombinant collagen articular cartilage repair gel and a preparation method thereof. Background Art

[0002] Osteoarthritis (OA) is one of the most common joint diseases with high recurrence and disability rate, which has placed a heavy burden on patients and society. Cartilage defect is one of the most common symptoms of OA. According to surveys, the prevalence of OA has a tendency to gradually increase with the aging of the population, and will become the main cause of disability in the world's population.

[0003] At present, the goal of drug treatment for cartilage damage and osteoarthritis is still to relieve pain and relieve symptoms. Drug treatment for cartilage damage and arthritis mainly includes: 1) injection of sodium hyaluronate lubricant; 2) oral administration of glucosamine and chondroitin sulfate, etc.; 3) use of non-steroidal anti-inflammatory drugs and other methods. In addition, local administration and systemic administration are the two routes of administration currently used for bone and joint treatment. Compared with systemic administration, OA patients can obtain higher local drug concentrations through intra-articular injection, thereby reducing adverse reactions. However, local injection of sodium hyaluronate cannot promote chondrocyte regeneration and intrinsic repair, and cannot promote the formation of fibrocartilage. Its main function is joint cavity lubrication. After oral administration, glucosamine and other drugs enter the human digestive system and are degraded into glucose, amino acids, etc., and cannot reach the joints in their original form to play a role. Collagen is the main component of connective tissues such as animal and human skin, blood vessels, bones, tendons, teeth and cartilage.

[0004] Studies have found that the destruction of the collagen fiber network and the loss of proteoglycans in the cartilage matrix lead to the degeneration and necrosis of chondrocytes, from shallow to deep, and eventually the disappearance of the entire cartilage layer and the proliferation of subchondral bone. The continuous degradation of collagen in cartilage leads to cartilage destruction and joint degeneration. Injecting type II collagen into the joint cavity in the early stage of cartilage injury and supplementing exogenous type II collagen for cartilage tissue will help alleviate the injury process and promote cartilage self-repair, thereby achieving radical treatment of the injury and avoiding joint degeneration causing arthritis.

[0005] However, currently, type II collagen is mainly obtained through animal tissue extraction and is limited to small-scale laboratory research. There are almost no raw materials that can be truly implemented and meet the quality requirements of non-toxic, non-pyrogenic, sterile, highly active and high-purity. With the rapid development of genetic engineering technology, the use of synthetic biology technology to obtain recombinant type II humanized collagen has broad application prospects.

[0006] Compared with natural collagen extracted from animals, recombinant humanized type II collagen has low antigenicity, which completely eliminates the inevitable virus risks of animal-derived materials and ensures the safety of gel materials in clinical use. There are domestic literature reports on the use of type II collagen for articular cavity injection for the treatment of cartilage defects and arthritis, but there are no research reports on the preparation of non-immunogenic, non-toxic, non-pyrogenic, and highly active micro-cross-linked articular cartilage repair gels using recombinant humanized type II collagen and other polysaccharide excipients. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a micro-crosslinked recombinant collagen articular cartilage repair gel and a preparation method thereof.

[0008] To solve the above problems, the technical solution adopted by the present invention is:

[0009] On the one hand, a micro-cross-linked recombinant collagen articular cartilage repair gel is provided, which is obtained by chemically cross-linking recombinant type II humanized collagen, sodium hyaluronate and chondroitin sulfate through EDC / NHS;

[0010] Among them, the raw materials include, by weight: 8-20 parts of recombinant humanized type II collagen, 0.5-2 parts of sodium hyaluronate, and 0.3-1 parts of chondroitin sulfate.

[0011] On the other hand, a method for preparing a micro-cross-linked recombinant collagen articular cartilage repair gel is provided, the method comprising:

[0012] Step S1, preparing recombinant type II humanized collagen using biological genetic engineering technology;

[0013] Step S2, mixing recombinant humanized type II collagen, sodium hyaluronate and chondroitin sulfate to prepare a mixed solution, and adding a crosslinking agent solution to the mixed solution to obtain a slightly crosslinked injectable gel;

[0014] Step S3, dialyzing the injectable gel to remove the cross-linking agent to obtain a dialyzed gel;

[0015] Step S4, sterilize the dialyzed gel with high temperature and high pressure wet heat, and then aseptically fill it into a pre-filled syringe to obtain a micro-cross-linked recombinant collagen articular cartilage repair gel.

[0016] As one embodiment of the invention, the amino acid sequence of the recombinant humanized type II collagen is shown as SEQ ID No.1.

[0017] As one embodiment of the invention, the nucleotide sequence of the recombinant human type II collagen is shown as SEQ ID No.2.

[0018] As an embodiment of the invention, in step S1, the method for preparing recombinant type II humanized collagen specifically comprises the following steps:

[0019] (1) Protein sequence design

[0020] Based on human type II collagen, an amino acid sequence as shown in SEQ No. 1 was designed;

[0021] (2) Gene design and synthesis

[0022] Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type II collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type II collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type II collagen fragment;

[0023] (3) Construction of expression vector:

[0024] The expression plasmid was obtained by ligating to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites;

[0025] (4) Construction and screening of expression strains:

[0026] The plasmid described in step (3) is transferred into the host cell competent state by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain;

[0027] (5) Inducible expression

[0028] Inducing the expression strain obtained in step (4) to express, and collecting the bacterial solution;

[0029] (6) Purification

[0030] The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a recombinant human type II collagen freeze-dried product.

[0031] As one embodiment of the invention, the host cell is Escherichia coli BL21 (DE3).

[0032] As an embodiment of the invention, step S2 includes:

[0033] Step S201, adding recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate to a 0.01M PBS solution cooled to below 30° C. to prepare a mixed solution; wherein, in the mixed solution, the concentration of the recombinant type II humanized collagen is 80-200 mg / mL, the concentration of sodium hyaluronate is 5-20 mg / mL, and the concentration of the chondroitin sulfate is 3-10 mg / mL;

[0034] Step S202, adding EDC / NHS crosslinker solution dropwise while stirring the mixed solution, and continuing to stir the reaction for 3-5 minutes after the addition is completed, and then placing the reaction solution at 4°C for crosslinking for 48 hours to obtain a slightly crosslinked injectable gel; wherein, as EDC, the EDC / NHS concentration is 0.2%, and the mass ratio of EDC to NHS is 1:1-1:4.

[0035] As an embodiment of the invention, step S3 comprises: dialyzing the injectable gel with 0.1M PBS at a low temperature of 4-8°C, replacing the dialysate every 2 hours, and dialyzing 5 times to remove the cross-linking agent.

[0036] As an embodiment of the invention, step S4 includes:

[0037] Step S401, sterilize the dialyzed gel by high temperature and high pressure wet heat, the sterilization condition is 121° C. for 20 minutes, and the gel state after sterilization is that it can be squeezed out through an injection needle;

[0038] Step S402, aseptically filling the gel into a pre-filled syringe to obtain the micro-cross-linked recombinant collagen articular cartilage repair gel.

[0039] The beneficial effects of adopting the above technical solution are:

[0040] The micro-crosslinked recombinant collagen articular cartilage repair gel provided by the present invention, wherein the recombinant type II humanized collagen has the excellent properties of natural collagen, and has no virus hidden dangers and immunogenicity; sodium hyaluronate is the main component of the synovial fluid in the joint cavity, and is also one of the components of the cartilage matrix, which plays a lubricating role in the joint and reduces the friction between the tissues; chondroitin sulfate has the effect of relieving pain and promoting cartilage regeneration. These three materials are prepared into a micro-crosslinked repair gel by chemical crosslinking, which has biocompatibility and degradability. Compared with simple hyaluronic acid, the viscosity increases after micro-crosslinking, effectively lubricates the joint, retains a long time at the cartilage defect site, maintains a certain degradation rate, and promotes the synthesis and secretion of endogenous collagen and hyaluronic acid, and the injection frequency is greatly reduced; in addition, the gel is applied to the rabbit cartilage defect model, indicating that the micro-crosslinked recombinant collagen articular cartilage repair gel has a good repair effect on cartilage defects, and has great potential and application value in the field of biomaterials.

[0041] During the preparation process of the gel provided by the present invention, no toxic substances remain, and the raw materials used are non-biologically toxic, so the prepared gel has good biocompatibility. At the same time, the gel also has good biodegradability, has the effect of inducing osteogenesis, and can promote the repair of bone defects.

[0042] The method for preparing recombinant humanized type II collagen provided by the present invention has raw materials without infectious sources of animal origin, has high biological safety, and can be widely used in the fields of medicine, medical devices, biomaterials, tissue engineering, cosmetics, etc.; the selected sequence has RGD and GAPGER integrin binding sites, which can promote the adhesion of cells to the recombinant human type II collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure is the electrophoresis pattern of the recombinant humanized type II collagen of the present invention.

[0044] Figure 2 The present invention is a flow chart for preparing the micro-cross-linked recombinant collagen articular cartilage repair gel.

[0045] Figure 3 It is an injectable form of the micro-cross-linked recombinant collagen articular cartilage repair gel of the present invention.

[0046] Figure 4 The present invention demonstrates the therapeutic effect of the micro-cross-linked recombinant collagen articular cartilage repair gel on cartilage defects in rabbits.

[0047] Figure 5 This is the expression of Col II in the rabbit cartilage defect repair model. DETAILED DESCRIPTION

[0048] 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.

[0049] Preparation Example 1

[0050] A method for preparing recombinant type II humanized collagen, the method comprising:

[0051] (1) Protein sequence design

[0052] According to the sequence characteristics of human type II collagen (UniProtKB / Swiss-Prot: P02458.3), the collagen sequence HC72 was designed, and its amino acid sequence is shown in SEQ ID NO.1.

[0053] (2) Gene design and synthesis

[0054] ①Gene design

[0055] Its N-terminus starts with methionine to reduce the degradation rate of the protein; a histidine tag is added to facilitate identification and purification. The selected sequence contains integrin binding sites: 2 RGD and 1 GAPGER. The amino acid sequence of the recombinant human type II collagen fragment is as follows:

[0056] SEQ ID NO.1

[0057] MHHHHHHGDRGDVGEKGPEGAPGKDGGRGLTGPIGPPGPAGANGEKGEVGPPGPAGSAGARGAPGERGETGPPGPAGFAGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGP KGARGAQGPPGATGFPGAAGRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAP

[0058] 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 E. coli expression. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human type II collagen was obtained, and the nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2:

[0059] ATGCACCACCATCACCACCATGGGGATAGGGGAGACGTTGGTGAGAAAGGTCCGGAAGGCGCTCCGGGCAAAGACGGCGGTCGTGGTTTGACCGGTCCGATTGGCCCGCCGGGCCCGGCGGGCGCGAACGGTGAGAAGGGCGAAGTTGGTCCGCCGGGTCCGGCGGGTTCTGCAGGTGCTCGCGGCGCGCCGGGCGAGCGCGGTGAAACCGGTCCGCCGGGCCCGGCGGGCTTCGCCGGCCCGCCGGGCGCTGATGGTCAGCCGGGCGCCAAAGGCGAGCAGGGTGAAGCAGGCCAAAAAGGTGATGCGGGTGCGCCAGGTCCGCAGGGCCCGTCCGGTGCGCCTGGCCCGCAAGGTCCAACGGGGGTGACTGGTCCGAAAGGTGCGCGTGGTGCGCAGGGTCCGCCGGGTGCTACCGGTTTTCCGGGCGCGGCAGGTAGAGTCGGTCCACCGGGCAGCAATGGAAACCCGGGCCCTCCGGGTCCGCCTGGCCCGTCGGGCAAGGACGGCCCCAAGGGTGCCCGTGGCGACAGCGGTCCACCGGGACGCGCAGGCGAACCGGGTCTGCAGGGCCCGGCGGGCCCGCCGGGCGAGAAGGGTGAGCCGGGTGATGATGGTCCGAGCGGTGCTGAAGGTCCGCCAGGCCCGCAAGGTCTGGCAGGTCAACGTGGGATCGTGGGCCTGCCGGGTCAACGTGGTGAACGTGGTTTCCCGGGCCTTCCGGGACCGAGCGGCGAGCCGGGTAAGCAGGGCGCGCCATAA

[0060] ②, Gene synthesis:

[0061] According to the above nucleic acid sequence, GenScript Biotech Corporation synthesized a nucleic acid fragment encoding a recombinant human type II collagen fragment.

[0062] (3) Construction of expression vector pET30a(+)-HC72

[0063] The obtained nucleic acid fragment SEQ ID NO.2 was ligated to the pET30a(+) plasmid (via the NdeI and XhoI multiple cloning sites) to obtain the recombinant plasmid pET30a(+)-HC72.

[0064] (4) Construction of expression strain BL21(DE3) / pET30a(+)-HC72

[0065] The construction of the expression strain refers to the method described in the Molecular Cloning Experiment Guide (3rd Edition) (written by J. Sambrook et al.), and the specific steps are as follows: pick a single colony of Escherichia coli BL21 (DE3) and inoculate it into an LB test tube, then culture it at 37°C overnight with shaking; add 0.5mL of the overnight culture solution to a conical flask containing 50mL LB, and culture it at 37°C with vigorous shaking for about 2 hours to allow the bacteria to grow to the early logarithmic phase; transfer the bacteria to an ice-cold 50mL polypropylene tube under sterile conditions and place it on ice for 10 minutes; centrifuge at 4°C, 4000rpm, pour out the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible; add 6mL of ice-cold 0.1mol / LCaCl 2 Resuspend the precipitate and place on ice for 30 minutes; centrifuge at 3000 rpm at 4°C, pour out the supernatant, invert the tube to allow the residual liquid to flow out as much as possible; add 1.2 mL of ice-cold 0.1 mol / L CaCl 2 Resuspend the pellet (if you want to prepare competent cells for storage at -70°C, add 0.1 mol / L CaCl containing 20% ​​glycerol). 2 Suspended bacteria), after placing at 4℃ for 5~24 hours, aspirate 200μl competent cell suspension, add the prepared recombinant plasmid pET30a(+)-HC72 (volume <10μl, DNA <50ng), mix gently, and place on ice for 30 minutes; heat shock in a 42℃ water bath for 90 seconds, and immediately place on ice for cooling; add 500μl liquid LB culture medium, mix well, and place in a 37℃ shaker for low-speed shaking to recover for 45 minutes (it can also be directly placed in a 37℃ water bath for recovery for 1 hour after adding LB, and the tube is shaken in the middle to suspend the cells); aspirate the transformed cells and spread them on a plate with antibiotics (kanamycin), place it in a 37℃ incubator and invert it for culture, and the colonies that grow are the expression strain BL21(DE3) / pET30a(+)-HC72.

[0066] (5) Induced expression of expression strain BL21(DE3) / pET30a(+)-HC72

[0067] A single colony of the prepared expression strain BL21(DE3) / pET30a(+)-HC72 was picked and placed in LB liquid medium containing 50 μg / mL Kan. After culturing overnight at 37°C and 200 r / min, it became an activated seed. Then, it was inoculated into a 5L fermenter filled with 3LCM medium at a 3% inoculum. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. When OD 600 When the expression reached 60, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM to induce expression, and the culture was continued for 12 hours, and the cells were collected by centrifugation.

[0068] (6) Purification of recombinant human type II collagen HC72

[0069] The theoretical isoelectric point of HC72 is 6.31, and the pH of the buffer solution is 8.0. The specific purification steps are as follows:

[0070] ①, breaking bacteria

[0071] The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis solution (20 mM Tris 500 mM NaCl, 20 mM imidazole, pH 8.0), with the ratio (m / v) of bacterial cells to lysis solution being 1:5-1:20, and 1:10 was selected in this example. The bacterial cells were ruptured using a high-pressure homogenizer. The ruptured bacterial solution was centrifuged at 12000 g for 1 h, and the supernatant was collected and filtered using a 0.45 μm filter membrane.

[0072] ②Affinity chromatography

[0073] a. Column balance:

[0074] The Ni Sepharose FF column was equilibrated with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0) until the baseline was stable.

[0075] b. Loading:

[0076] The supernatant was added to the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0077] c. Column flushing:

[0078] Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0079] d. Elution of target protein:

[0080] The solution was eluted with 80% Buffer A and 20% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type II collagen HC72.

[0081] ③. Ion exchange chromatography

[0082] a. Sample processing:

[0083] The affinity chromatography eluate was diluted with ion exchange solution A (20 mM Tris, pH 8.0) or ultrafiltered to a conductivity of less than 5 mS / cm.

[0084] b. Column balance:

[0085] The Q Sepharose FF column was equilibrated with buffer A until the baseline was stable.

[0086] c. Loading:

[0087] The renaturation solution was added to the chromatography column, and the column retention time was controlled to be no less than 5 minutes.

[0088] d. Column flushing:

[0089] Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0090] e. Target protein elution:

[0091] The solution was eluted with 75% Buffer A and 25% Buffer B (20 mM Tris, 1 M NaCl, pH 8.0), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type II collagen HC72.

[0092] ④ Desalination

[0093] The HC72 solution was replaced with purified water by equal volume replacement using a 5 kDa ultrafiltration membrane device to remove salt from the solution, and then filtered with a 0.22 μm filter membrane and freeze-dried to obtain a recombinant collagen freeze-dried product. Figure 1 The electrophoretic pattern of recombinant humanized type II collagen is shown.

[0094] Example 1

[0095] A method for preparing a micro-cross-linked recombinant collagen articular cartilage repair gel, the method comprising:

[0096] 1. Preparation of micro-cross-linked injectable gel

[0097] (1) The recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate obtained in Preparation Example 1 were added to a 0.01 M PBS solution to prepare a mixed solution; in the mixed solution, the concentration of recombinant type II humanized collagen was 150 mg / mL, the final concentration of sodium hyaluronate was 12 mg / mL, and the final concentration of chondroitin sulfate was 6 mg / mL;

[0098] (2) Add EDC / NHS crosslinker solution dropwise while stirring the mixed solution. After the addition is completed, continue to stir the reaction for 3 to 5 minutes. Then, place the reaction solution at 4°C for crosslinking for 48 hours to obtain a slightly cross-linked injectable gel; wherein the mass percentage concentration of EDC is 0.2%, and the mass ratio of EDC to NHS is 1:2.

[0099] 2. Dialysis of injectable gel

[0100] The injectable gel was dialyzed with 0.1M PBS at 4-8°C, with the dialysate replaced every 2 hours. The dialyzate was dialyzed 5 times to remove the cross-linking agent, thereby obtaining a dialyzed gel.

[0101] 3. Preparation of micro-cross-linked recombinant collagen articular cartilage repair gel

[0102] (1) The dialysis gel is sterilized by high temperature and high pressure wet heat sterilization at 121°C for 20 minutes. After sterilization, the gel is in a state that can be squeezed out through an injection needle.

[0103] (2) Aseptically filling the gel into a pre-filled syringe to obtain the micro-cross-linked recombinant collagen articular cartilage repair gel. The injectable form of the obtained micro-cross-linked recombinant collagen articular cartilage repair gel is as follows: Figure 3 shown.

[0104] Example 2

[0105] A method for preparing a micro-cross-linked recombinant collagen articular cartilage repair gel, the method comprising:

[0106] 1. Preparation of micro-cross-linked injectable gel

[0107] (1) The recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate obtained in Preparation Example 1 were added to a 0.01 M PBS solution to prepare a mixed solution; in the mixed solution, the concentration of recombinant type II humanized collagen was 80 mg / mL, the final concentration of sodium hyaluronate was 20 mg / mL, and the final concentration of chondroitin sulfate was 3 mg / mL;

[0108] (2) Add EDC / NHS crosslinker solution dropwise while stirring the mixed solution. After the addition is completed, continue to stir the reaction for 3 to 5 minutes. Then, place the reaction solution at 4°C for crosslinking for 48 hours to obtain a slightly cross-linked injectable gel; wherein the mass percentage concentration of EDC is 0.2%, and the mass ratio of EDC to NHS is 1:1.

[0109] 2. Dialysis of injectable gel

[0110] The injectable gel was dialyzed with 0.1M PBS at 4-8°C, with the dialysate replaced every 2 hours. The dialyzate was dialyzed 5 times to remove the cross-linking agent, thereby obtaining a dialyzed gel.

[0111] 3. Preparation of micro-cross-linked recombinant collagen articular cartilage repair gel

[0112] (1) The dialysis gel is sterilized by high temperature and high pressure wet heat sterilization at 121°C for 20 minutes. After sterilization, the gel is in a state that can be squeezed out through an injection needle.

[0113] (2) Aseptically fill the gel into a pre-filled syringe to obtain the micro-cross-linked recombinant collagen articular cartilage repair gel.

[0114] Example 3

[0115] A method for preparing a micro-cross-linked recombinant collagen articular cartilage repair gel, the method comprising:

[0116] 1. Preparation of micro-cross-linked injectable gel

[0117] (1) The recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate obtained in Preparation Example 1 were added to a 0.01 M PBS solution to prepare a mixed solution; in the mixed solution, the concentration of recombinant type II humanized collagen was 200 mg / mL, the final concentration of sodium hyaluronate was 5 mg / mL, and the final concentration of chondroitin sulfate was 10 mg / mL;

[0118] (2) Add EDC / NHS crosslinker solution dropwise while stirring the mixed solution. After the addition is completed, continue to stir the reaction for 3 to 5 minutes. Then, place the reaction solution at 4°C for crosslinking for 48 hours to obtain a slightly cross-linked injectable gel; wherein, EDC is used as the reference, the EDC / NHS concentration is 0.2%, and the mass ratio of EDC to NHS is 1:4.

[0119] 2. Dialysis of injectable gel

[0120] The injectable gel was dialyzed with 0.1M PBS at 4-8°C, with the dialysate replaced every 2 hours. The dialyzate was dialyzed 5 times to remove the cross-linking agent, thereby obtaining a dialyzed gel.

[0121] 3. Preparation of micro-cross-linked recombinant collagen articular cartilage repair gel

[0122] (1) The dialysis gel is sterilized by high temperature and high pressure wet heat sterilization at 121°C for 20 minutes. After sterilization, the gel is in a state that can be squeezed out through an injection needle.

[0123] (2) Aseptically fill the gel into a pre-filled syringe to obtain the micro-cross-linked recombinant collagen articular cartilage repair gel.

[0124] Comparative Example 1

[0125] Preparation of uncross-linked composite gel: Add the recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate obtained in Preparation Example 1 to a 0.01M PBS solution to prepare a composite gel; in the composite gel, the concentration of recombinant type II humanized collagen is 150 mg / mL, the final concentration of sodium hyaluronate is 12 mg / mL, and the final concentration of chondroitin sulfate is 6 mg / mL. The difference from Example 1 is that only the three components are compounded into a gel, and no micro-cross-linking reaction is performed. After moist heat sterilization, the viscosity is significantly reduced, the retention time at the cartilage defect site is greatly shortened, and the efficacy is also reduced.

[0126] Comparative Example 2

[0127] Preparation of micro-cross-linked articular cartilage repair gel without recombinant collagen: The preparation process is the same as that of Example 1, except that only sodium hyaluronate and chondroitin sulfate are cross-linked, and no recombinant type II humanized collagen component is contained.

[0128] Effect Example 1 In vitro cytotoxicity test

[0129] The concentration is 2×10 5 200 μl / well of L929 cell suspension with a concentration of 1 cell / mL was added to a 96-well plate and incubated in 5% CO 2 , incubate in a 37°C incubator for 6 hours and aspirate the culture medium. Replace the culture medium containing the gel extract of Example 1 (the volume ratio of gel to serum-free culture medium is 1:10) and continue to culture for 24 hours. Take out the 96-well plate and add CCK8 10μ1 / well. Place in an incubator and continue to culture for 2-4 hours depending on the display. Measure the absorbance at 450nm on an ELISA reader, and calculate the cell activity: cell activity = (OD value of the test well - OD value of the blank control well) / OD value of the negative control well.

[0130] The results of the cytotoxicity test showed that the cell survival rate treated with the gel extract prepared in Example 1 was not significantly different from that of the blank control group, and the survival rate was above 90%, indicating that the gel had no cytotoxicity and good biocompatibility.

[0131] Effect Example 2 Cell Proliferation and Differentiation Test

[0132] BMSCs were inoculated in DMEM / F12 culture medium (containing 10% FBS and 100 U / mL double antibody) and incubated at 37°C with 5% CO 2 The BMSCs were then inoculated into the gels of Example 1, Comparative Example 1, and Comparative Example 2 (cell inoculation concentration 1×10 6 Cells / mL) were added, and the culture medium was changed to chondrogenic differentiation medium after 24 hours of culture. The cells were cultured for 1 day, 3 days, and 7 days, respectively, and the cell proliferation and chondrogenic differentiation ability were detected. The cell proliferation level was evaluated by CCK8 method, and the results are shown in Table 1; the expression of chondrogenic differentiation marker Col II was detected by Western blot, and the results are shown in Table 2.

[0133] Table 1 Cell proliferation in each group of gels

[0134]

[0135] Table 2 Relative expression levels of cell chondrogenic differentiation marker Col II in each group of gels

[0136]

[0137] The results showed that the micro-cross-linked cartilage repair gels prepared in Examples 1 to 3 had a more obvious proliferation promoting effect and cartilage differentiation ability on BMSCs. As the action time prolonged, the effect of the micro-cross-linked gel group was more significant, indicating that the gel formed after micro-cross-linking of the three components can maintain a certain degradation rate and has a more lasting effect.

[0138] Effect Example 3 Rabbit articular cartilage defect repair test

[0139] A rabbit articular cartilage defect model was established. Mature male rabbits were selected for the experiment and anesthetized by injection of 10% chloral hydrate at a dose of 2.5 mL / kg. The rabbit hind limbs were then depilated, the knee joints were fixed, and disinfected with 75% iodine tincture. A scalpel was used to make an incision on the outside of the rabbit patella to expose the articular cartilage, and a dental drill was used to prepare a defect with a diameter of 3 mm and a depth of 1.5 mm in the articular cartilage. After the model was successfully established, the rabbits were divided into groups for treatment. The groups were as follows: the model group (M group, 3 rabbits) was not treated; the control group (HA group, 3 rabbits) was injected with 100 μl of HA solution at the defect; the experimental group (Example 1, Gel group, 3 rabbits) was injected with 100 μl of micro-cross-linked recombinant collagen articular cartilage repair gel at the defect. After treatment, the fascia layer was sutured continuously, the skin was sutured intermittently, and disinfection was performed. Two days after surgery, the rabbits received intramuscular injections of the antibiotic ceftriaxone sodium to prevent infection. Six weeks after surgery, the articular cartilage repair was evaluated, and the results are as follows. Figure 4 and Figure 5 As shown. Figure 4and Figure 5 It can be seen that the gel of the present invention has a good repairing effect on rabbit articular cartilage defects, and the relative expression level of endogenous type II collagen, a cartilage differentiation marker, is significantly increased and higher than that of the control group.

Claims

1. A micro-cross-linked recombinant collagen articular cartilage repair gel, characterized in that: It is obtained by cross-linking recombinant humanized type II collagen, sodium hyaluronate and chondroitin sulfate through EDC / NHS chemical cross-linking; Among them, the raw materials include, by weight: 8-20 parts of recombinant humanized type II collagen, 0.5-2 parts of sodium hyaluronate, and 0.3-1 parts of chondroitin sulfate. The amino acid sequence of the recombinant humanized type II collagen is shown in SEQ ID No.

1.

2. A method for preparing a micro-cross-linked recombinant collagen articular cartilage repair gel, characterized in that: Methods include: Step S1, preparing recombinant type II humanized collagen by using biological genetic engineering technology; the amino acid sequence of the recombinant type II humanized collagen is shown in SEQ ID No. 1; Step S2, mixing recombinant humanized type II collagen, sodium hyaluronate and chondroitin sulfate to prepare a mixed solution, and adding a crosslinking agent solution to the mixed solution to obtain a slightly crosslinked injectable gel; Step S3, dialyzing the injectable gel to remove the cross-linking agent to obtain a dialyzed gel; Step S4, sterilize the dialyzed gel with high temperature and high pressure wet heat, and then aseptically fill it into a pre-filled syringe to obtain a micro-cross-linked recombinant collagen articular cartilage repair gel.

3. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 2, characterized in that: The nucleotide sequence of the recombinant human type II collagen is shown in SEQ ID No.

2.

4. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 2, characterized in that: In step S1, the method for preparing recombinant type II humanized collagen specifically comprises the following steps: (1) Protein sequence design Based on human type II collagen, an amino acid sequence as shown in SEQ No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type II collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type II collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type II collagen fragment; (3) Construction of expression vector: The expression plasmid was obtained by ligating to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites; (4) Construction and screening of expression strains: The plasmid described in step (3) is transferred into the host cell competent state by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Inducing the expression strain obtained in step (4) to express, and collecting the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a recombinant human type II collagen freeze-dried product.

5. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 4, characterized in that: The host cell is Escherichia coli BL21 (DE3).

6. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 2, characterized in that: Step S2 includes: Step S201, adding recombinant type II humanized collagen, sodium hyaluronate, and chondroitin sulfate to a 0.01M PBS solution cooled to below 30° C. to prepare a mixed solution; wherein, in the mixed solution, the concentration of the recombinant type II humanized collagen is 80-200 mg / mL, the concentration of sodium hyaluronate is 5-20 mg / mL, and the concentration of the chondroitin sulfate is 3-10 mg / mL; Step S202, adding EDC / NHS crosslinker solution dropwise while stirring the mixed solution, and continuing to stir the reaction for 3-5 minutes after the addition is completed, and then placing the reaction solution at 4°C for crosslinking for 48 hours to obtain a slightly crosslinked injectable gel; wherein, as EDC, the EDC / NHS concentration is 0.2%, and the mass ratio of EDC to NHS is 1:1-1:

4.

7. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 2, characterized in that: Step S3 comprises: dialyzing the injectable gel at a low temperature of 4-8° C. using 0.1 M PBS, replacing the dialysate once every 2 hours, and dialyzing for 5 times to remove the cross-linking agent.

8. The method for preparing a micro-crosslinked recombinant collagen articular cartilage repair gel according to claim 2, characterized in that: Step S4 includes: Step S401, sterilize the dialyzed gel by high temperature and high pressure wet heat, the sterilization condition is 121° C. for 20 minutes, and the gel state after sterilization is that it can be squeezed out through an injection needle; Step S402, aseptically filling the gel into a pre-filled syringe to obtain the micro-cross-linked recombinant collagen articular cartilage repair gel.

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