Collagen and chitosan cross-linked temperature-sensitive gel for repairing articular cartilage and preparation method of collagen and chitosan cross-linked temperature-sensitive gel

Abstract

The invention relates to the field of preparation of materials for articular cartilage repair, in particular to collagen and chitosan cross-linked temperature-sensitive gel for articular cartilage repair and a preparation method of the collagen and chitosan cross-linked temperature-sensitive gel. The cartilage repair material prepared by the invention adopts the recombinant human collagen with a triple helix structure as a main raw material. The raw material is prepared by using a mammalian cell expression system, is close to protein folding and polymerization of natural protein, and has a spatial structure and modification necessary for active protein. The recombinant human collagen with the triple-helix structure is good in biocompatibility and free of rejection reaction and anaphylactic reaction, and the risk of disease transmission possibly caused by animal-derived collagen can be avoided. The injection type temperature-sensitive gel is liquid at low temperature and is converted into hydrogel at 37 DEG C, so that the injection type temperature-sensitive gel is suitable for being used as a transfer carrier for in-vivo injection treatment, secondary injury of an organism is effectively avoided, and the injection type temperature-sensitive gel has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of preparation of materials for repairing articular cartilage, and in particular to a collagen chitosan cross-linked thermosensitive gel for repairing articular cartilage and a preparation method thereof. Background Art

[0002] Knee arthritis is a degenerative disease that primarily affects middle-aged and elderly individuals. Symptoms include redness, swelling, and pain in the knee, pain climbing stairs, and knee soreness and discomfort when sitting, standing, or walking. The patient population is large. There are few cures for knee arthritis, and some patients often end up having to undergo knee replacement surgery.

[0003] Currently, there are many ways to relieve the pain of knee arthritis, mainly conservative treatment and arthroscopic minimally invasive treatment. These two methods have their own advantages and disadvantages, and the long-term clinical benefits of these two therapies are limited. For example, conservative treatment often involves injecting corticosteroids, sodium hyaluronate, etc. into the joint cavity. Although injections of corticosteroids, sodium hyaluronate, etc. can have anti-inflammatory, pain-relieving, lubricating, and improving functional activities, long-term injections of corticosteroids into the joint cavity can lead to cartilage metabolism imbalance and osteoporosis; injections of hyaluronic acid into the joint cavity can lubricate the joints and relieve symptoms such as pain, but cannot repair cartilage damage or delay the progression of knee osteoarthritis (KOA).

[0004] Therefore, the invention of a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair has great clinical application prospects. Summary of the Invention

[0005] In response to the above-mentioned existing technical problems, the present invention aims to invent a collagen chitosan cross-linked thermosensitive gel for articular cartilage repair and a preparation method. The preparation method of this cross-linked thermosensitive gel is simple. The injectable thermosensitive gel is liquid at low temperatures and converts into a hydrogel at 37°C. It is suitable as a delivery carrier for in vivo injection therapy, effectively avoiding secondary damage to the body, and therefore has broad clinical application prospects.

[0006] The present invention discloses a method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, comprising the following steps:

[0007] (1) Weigh an appropriate amount of recombinant human collagen solution, add a pH adjuster to adjust the pH to 7, add methacrylic anhydride for modification, let it stand at 4°C, then add a separating agent, and centrifuge to obtain the collagen precipitate;

[0008] (2) adding lysine oxidase to the collagen precipitate obtained in step (1) under stirring, mixing and cross-linking at 25° C., then transferring to an ultra-low temperature refrigerator and freezing for 4 h, transferring the frozen sample to a vacuum freeze dryer, and vacuum freeze-drying for 48 h to obtain a collagen sponge;

[0009] (3) preparing a sodium hyaluronate solution, adding methacrylic anhydride to the sodium hyaluronate solution under ice bath and stirring conditions to prepare a sodium hyaluronate thermosensitive gel, and adding the collagen sponge to mix evenly;

[0010] (4) converting the modified collagen sodium hyaluronate thermosensitive gel solution obtained in step (3) into a viscous gel at 32-37°C.

[0011] Preferably, the concentration of the recombinant human collagen solution in step (1) is 1-2.5 mg / ml.

[0012] Preferably, in step (1), the mass ratio of the recombinant human collagen solution to methacrylic anhydride is (7-20):1.

[0013] Preferably, in step (1), the pH adjuster comprises sodium hydroxide, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0014] Preferably, the standing time at 4°C in step (1) is 12 to 24 hours; and the cross-linking time at 25°C in step (2) is 6 to 12 hours.

[0015] Preferably, in step (1), the separating agent comprises ethanol, propylene glycol or glycerol.

[0016] Preferably, in step (2), the mass ratio of the recombinant human collagen precipitate to lysine oxidase is (15-50):1.

[0017] Preferably, the molecular weight of the sodium hyaluronate in step (3) is 100,000 to 250,000 Da.

[0018] Preferably, in step (3), the concentration of the sodium hyaluronate solution is 1-4 mg / ml; the mass ratio of the sodium hyaluronate solution to methacrylic anhydride is (18-25):1.

[0019] The collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair is prepared by any of the above methods for preparing the collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The purpose of the present invention is to provide a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair and a preparation method thereof. Compared with the prior art, the cross-linked thermosensitive gel prepared by the present invention has the following advantages:

[0022] (1) In clinical use, it can be implanted through arthroscopic minimally invasive surgery and directly solidified in situ in the cartilage defect. The surgery is minimally invasive and takes a short time, which is in line with the minimally invasive concept of orthopedic treatment.

[0023] (2) After implantation, the material can solidify in situ in the cartilage defect to form an ordered collagen fiber structure, giving the material higher degradation resistance and mechanical strength.

[0024] (3) There is no need for a secondary surgery on the cartilage defect site, and it can be applied to the repair of cartilage defects of any characteristics.

[0025] (4) It is well integrated with the surrounding area of the cartilage defect and can induce the growth of chondrocytes around the defect, promote chondrocyte proliferation, and secrete type II collagen, thereby achieving true cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Reaction formula for modification of collagen particles.

[0027] Figure 2 : Schematic representation of the appearance of Examples 1 to 6 and Comparative Examples 1 to 3. From top to bottom and from left to right, the appearances of Examples 1 to 6 and Comparative Examples 1 to 3 are shown.

[0028] Figure 3 : Schematic diagram of the substitution degree calculation formula.

[0029] Figure 4 : Schematic diagram of identification results. The solid line represents the stretching vibration peak of collagen before modification; the dotted line represents the stretching vibration peak of collagen after modification.

[0030] Figure 5 : Schematic diagram of animal experiment results. DETAILED DESCRIPTION

[0031] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0032] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0033] Example 1: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair, comprising the following steps:

[0034] (1) Weigh 2 mg / ml recombinant human collagen solution, add sodium hydroxide to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 10:1 to the recombinant human collagen solution, stand at 4°C for 16 h to modify the collagen, add ethanol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0035] (2) adding a cross-linking agent lysine oxidase at a mass ratio of 30:1 to the collagen precipitate obtained in step (1) under stirring, mixing, cross-linking at 25°C for 12 hours, and then transferring to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0036] (3) Prepare a 2 mg / ml sodium hyaluronate solution with a molecular weight of 200,000 Da. Add methacrylic anhydride at a mass ratio of 20:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0037] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0038] Example 2: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair, comprising the following steps:

[0039] (1) Weigh 1.5 mg / ml recombinant human collagen solution, add disodium hydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 8:1 to the recombinant human collagen solution, let it stand at 4°C for 14 h to modify the collagen, add ethanol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0040] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 25:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 12 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0041] (3) Prepare a 3 mg / ml sodium hyaluronate solution with a molecular weight of 150,000 Da. Add methacrylic anhydride at a mass ratio of 18:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0042] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0043] Example 3: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair, comprising the following steps:

[0044] (1) Weigh 2.5 mg / ml recombinant human collagen solution, add potassium dihydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 12:1 to the recombinant human collagen solution, let it stand at 4°C for 18 h to modify the collagen, add propylene glycol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0045] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 15:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 14 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0046] (3) Prepare a 1.5 mg / ml sodium hyaluronate solution with a molecular weight of 250,000 Da. Add methacrylic anhydride at a mass ratio of 20:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0047] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0048] Example 4: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, comprising the following steps:

[0049] (1) Weigh 1.8 mg / ml recombinant human collagen solution, add disodium hydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 7:1 to the recombinant human collagen solution, let it stand at 4°C for 20 h to modify the collagen, add glycerol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0050] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 28:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 10 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0051] (3) Prepare a 2 mg / ml sodium hyaluronate solution with a molecular weight of 200,000 Da. Add methacrylic anhydride at a mass ratio of 20:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0052] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0053] Example 5: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, comprising the following steps:

[0054] (1) Weigh 1.2 mg / ml recombinant human collagen solution, add sodium hydroxide to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 14:1 to the recombinant human collagen solution, let it stand at 4°C for 24 hours to modify the collagen, add ethanol, and centrifuge to obtain the collagen precipitate.

[0055] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 35:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 8 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0056] (3) Prepare a 4 mg / ml sodium hyaluronate solution with a molecular weight of 120,000 Da. Add methacrylic anhydride at a mass ratio of 20:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add the collagen sponge to the solution and mix evenly.

[0057] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0058] Example 6: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, comprising the following steps:

[0059] (1) Weigh 1.0 mg / ml recombinant human collagen solution, add disodium hydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 20:1 to the recombinant human collagen solution, let it stand at 4°C for 12 hours to modify the collagen, add ethanol, and centrifuge to obtain the collagen precipitate.

[0060] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 50:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 6 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0061] (3) Prepare a 1 mg / ml sodium hyaluronate solution with a molecular weight of 100,000 Da. Add methacrylic anhydride at a mass ratio of 25:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0062] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0063] Comparative Example 1: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair, comprising the following steps:

[0064] (1) Weigh 0.2 mg / ml recombinant human collagen solution, add disodium hydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 30:1 to the recombinant human collagen solution, let it stand at 4°C for 3 h to modify the collagen, add ethanol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0065] (2) Lysine oxidase, a crosslinking agent, was added to the collagen precipitate at a mass ratio of 60:1 under stirring. The mixture was then crosslinked at 25°C for 3 h. The mixture was then transferred to an ultra-low temperature freezer and frozen for 4 h. The ultra-low temperature frozen sample was quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 h to obtain a collagen sponge.

[0066] (3) Prepare a 0.5 mg / ml sodium hyaluronate solution with a molecular weight of 100,000 Da. Add methacrylic anhydride at a mass ratio of 35:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0067] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0068] Comparative Example 2: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, comprising the following steps:

[0069] (1) Weigh 1.0 mg / ml recombinant human collagen solution, add sodium hydroxide to adjust the pH to 9.0, add methacrylic anhydride at a mass ratio of 20:1 to the recombinant human collagen solution, let it stand at 4°C for 12 h to modify the collagen, add ethanol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0070] (2) Add the cross-linking agent lysine oxidase at a mass ratio of 50:1 to the above collagen precipitate under stirring, mix well, cross-link at 25°C for 6 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0071] (3) Prepare a 1 mg / ml sodium hyaluronate solution with a molecular weight of 100,000 Da. Add methacrylic anhydride at a mass ratio of 25:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0072] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0073] Comparative Example 3: A method for preparing a collagen-chitosan cross-linked thermosensitive gel for articular cartilage repair, comprising the following steps:

[0074] (1) Weigh 1.0 mg / ml recombinant human collagen solution, add disodium hydrogen phosphate to adjust the pH to 7, add methacrylic anhydride at a mass ratio of 20:1 to the recombinant human collagen solution, let it stand at 4°C for 12 h to modify the collagen, add ethanol as a separating agent, and centrifuge to obtain the collagen precipitate.

[0075] (2) Add glutaraldehyde, a crosslinking agent, at a mass ratio of 50:1 to the above collagen precipitate under stirring, mix well, crosslink at 25°C for 6 hours, and then transfer to an ultra-low temperature refrigerator for freezing for 4 hours. The ultra-low temperature frozen sample is quickly transferred to a pre-cooled vacuum freeze dryer and vacuum freeze-dried for 48 hours to obtain a collagen sponge.

[0076] (3) Prepare a 1 mg / ml sodium hyaluronate solution with a molecular weight of 100,000 Da. Add methacrylic anhydride at a mass ratio of 25:1 to the sodium hyaluronate solution under ice bath and stirring to prepare a low-temperature sodium hyaluronate thermosensitive gel. Add collagen sponge to the solution and mix evenly.

[0077] (4) The low-temperature modified collagen sodium hyaluronate thermosensitive gel solution can quickly transform into a viscous gel when kept at 32-37°C.

[0078] The collagen chitosan cross-linked thermosensitive gels prepared in Examples 1 to 6 and Comparative Examples 1 to 3 were characterized, and the results are as follows:

[0079] 1. Appearance: Figure 2As shown, from top to bottom and from left to right, the appearances of Examples 1 to 6 and Comparative Examples 1 to 3 are respectively, all of which are white, milky white or slightly yellow viscous liquids without any foreign matter visible to the naked eye.

[0080] 2. Degree of substitution: Use the Col-MA detection method: prepare glycine into solutions with different concentration gradients and make a standard curve. Prepare a mixture of calcium acetate and magnesium acetate solids (CMA) into a 0.6-1 mg / mL CMA solution, then take 1 mL of the solution to be tested in a 5 mL brown volumetric flask, and add 1 mL of sodium bicarbonate buffer solution (pH 8.4) and 1 mL of 0.1% 2,4,6-trinitrobenzenesulfonic acid solution (TNBSA) to the brown volumetric flask. Place it in a water bath constant temperature oscillator, shake and incubate at 38°C for 2 hours, then take it out, add 1 mL (10%) of sodium dodecyl sulfate aqueous solution and 0.5 mL (1 mol / L) of hydrochloric acid solution to the brown volumetric flask. Then measure the absorbance of the solution at 345 nm to obtain the amino content of the modified collagen, and calculate the corresponding degree of substitution according to the formula. The formula is as follows: Figure 3 As shown in the formula (where A is the degree of substitution, %; N0 is the free amino group concentration of unmodified collagen, mmol / L; N f is the concentration of free amino groups in modified collagen, mmol / L); the degree of substitution should be in the range of 50%-80%; the test results are shown in Table 4.

[0081] 3. Identification: The peak of collagen before modification at 1030cm-1 is the CN stretching vibration peak of the primary amino group on the lysine residue. After modification, the peak of Col-MA at 1030cm-1 disappears, indicating that MA reacts with the amino group of lysine in collagen. Col-MA has a new peak at 925cm-1, which is the RC=CH2 out-of-plane bending (deformation) vibration peak. The C=C of the surface MA has been successfully modified on the collagen molecular chain; the results are as follows Figure 4 shown.

[0082] 4. Osmolality: Determined according to method 0632 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the osmolality should be within the range of 270 mOsmol / L-360 mOsmol / L. The test results are shown in Table 4.

[0083] 5. pH value: Determined according to method 0633 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the pH value should be within the range of 6.8-7.6; the test results are shown in Table 4.

[0084] 6. Dynamic viscosity: Determined according to method 0633 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), using a Brookfield cone-plate viscometer (USA). The test temperature was 25°C, with a CPA-52Z spindle and a speed of 10 rpm. The torque should be between 10% and 90%, and the dynamic viscosity should be between 5000 mPa﹒ s and 100,000 mPa﹒ s. The test results are shown in Table 4.

[0085] 7. Sterility: Sterility should be observed according to the method specified in 1101 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition). The test results are shown in Table 4.

[0086] 8. Bacterial endotoxins: Take 0.2 g of this product, add endotoxin test water at 0.2 g / mL (endotoxin content in the test water is less than 0.015 EU / mL), and extract by aseptic shaking at 37±1°C for 60 min at a shaking rate of 120 / min. Take the extract (particle-free) and use the Limulus amebocyte lysate with a sensitivity of 0.25 EU / mL. According to the method specified in 1143 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), the bacterial endotoxin should be less than 0.5 EU / mL. The test results are shown in Table 4.

[0087] 9. Cytotoxicity test: (The results of the cytotoxicity test are shown in Table 3)

[0088] (1) Sample preparation: Samples were extracted according to the following ratio (sample: extract volume) in a constant temperature shaker at 37°C and 60 rpm for 24 hours. After the extraction, the extract was checked for changes and used immediately in the experiment. The extract was not filtered, centrifuged, diluted, or otherwise subjected to pH adjustment. Blank, negative, and positive control samples were also prepared; as shown in Table 1:

[0089] Table 1 Sample preparation

[0090]

[0091] (2) Test method (sterile operation during the test): L-929 cells were cultured in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin, streptomycin) and cultured in a 37°C, 5% CO2 incubator. The cells were digested with trypsin to prepare a cell suspension, centrifuged (200G, 3min), and then the cells were redispersed in fresh culture medium to adjust the cell density to 1x105cells / ml of cell suspension; the above cell suspension was inoculated into 96-well culture plates, 100μL per well, and cultured in a carbon dioxide incubator for 24 hours (5% CO2, 37°C, humidity>90%); after the cells grew into a monolayer, the original culture medium was aspirated, and 100μL of different concentrations of test samples (100%, 75%, 50%, 25%), blank control solution, positive control solution (100%) and negative control solution (100%) were added respectively, and the plates were placed at 37°C, 5% CO2 culture for 24 hours, with 6 parallel samples for each group; after 24 hours of culture, the 96-well plate was removed, and the cell morphology was observed under a microscope. Then, the liquid was aspirated, and 50 μL (1 mg / ml) of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well. The cells were cultured in a CO2 incubator for 2 hours, the MTT solution was discarded, and 100 μL of isopropanol solution was added to each well. The plate was shaken, and the absorbance was measured at 570 nm on a microplate reader (reference wavelength 650 nm).

[0092] Table 2 Cell morphology description

[0093]

[0094] (3) Evaluation criteria: When the survival rate is low, the test sample has a higher potential cytotoxicity; if the survival rate drops to <70% of the blank, it has potential cytotoxicity; the survival rate of the 50% extract of the test sample should be at least the same as or higher than the survival rate of the 100% extract, otherwise the test should be repeated; the survival rate (%) of the 100% test sample extract is the final result.

[0095] Table 3 Cytotoxicity test results

[0096]

[0097]

[0098] Table 4 Test results of examples and comparative examples

[0099]

[0100] Table 3, Table 4, Figure 2 and Figure 4The test results show that the substitution degrees of Examples 1 to 6 all meet the standards (55%-75%), indicating that methacrylic anhydride (MA) reacts fully with the amino groups of collagen to form a stable Col-MA structure. The substitution degrees of Comparative Examples 1 to 3 are significantly lower (25%-30%) because the collagen concentration is too low (0.2 mg / ml) and the reaction time is insufficient (3 h), resulting in insufficient modification.

[0101] The infrared spectra of Examples 1-6 and Comparative Examples 1 and 3 showed the disappearance of the MA characteristic C=C peak (925 cm-1) and the lysine residue peak (1030 cm-1), confirming that MA successfully modified collagen. Comparative Example 2 failed the identification because the high pH (9.0) destroyed the collagen structure and affected the exposure of the reaction sites.

[0102] The osmotic pressure (285-299 mOsmol / L) and pH (7.06-7.21) of Examples 1 to 6 all meet the requirements of the Pharmacopoeia and have good biocompatibility; the pH value of Comparative Example 2 is relatively high (8.80).

[0103] The viscosities of Examples 1-5 met the standard (39,000-72,000 mPa·s) and were suitable for injection. The lower viscosity of Example 6 (39,000 mPa·s) was due to the low collagen concentration (1.0 mg / ml) and cross-linker ratio (50:1), resulting in a loose network structure. The viscosities of Comparative Examples 1-3 were significantly lower (20,000-30,000 mPa·s), making it impossible to maintain gel morphological stability.

[0104] All samples of Examples 1 to 6 and Comparative Examples 1 to 3 were sterile and met the endotoxin standard (<0.5 EU / mL), meeting the safety requirements for implant materials.

[0105] The cell viability of Examples 1-6 (83.3%-87.8%) was >70%, indicating no potential toxicity. The cell viability of Comparative Examples 2-3 decreased significantly (5.6%-30%). Comparative Example 2 was due to cell damage caused by abnormal pH, and Comparative Example 3 was due to residual toxicity of the cross-linking agent glutaraldehyde.

[0106] 10. Animal Experiment: Rats were used as an animal model, and the photocurable cross-linked collagen gel obtained in Example 1 was used as the material. The rats were placed in a supine position on the operating table with their limbs immobilized. Anesthesia was administered with an intraperitoneal injection of 10% chloral hydrate (0.1 ml / 20 g). The anesthesia lasted for approximately 1 hour. If the operation lasted longer than 1 hour, additional anesthetic was administered during the operation to enhance the anesthesia effect. The rats were placed in a supine position on the operating table, with their forelimbs immobilized. The legs were separated, leaving only the knee joint free. A longitudinal incision was made in the skin anterior to the right knee from the medial side of the patellar ligament. The subcutaneous tissue and surrounding free tissue were peeled off. The retinaculum and joint capsule were incised 1 mm from the medial edge of the patella, and the surrounding tissue was peeled off. The patella was then dislocated laterally, and the knee was flexed to expose the femoral trochlea. A 1 mm diameter drill was used to create a cartilage defect with a diameter of 1 mm and a depth of 1 mm in the center of the femoral trochlea. Hemostasis was achieved. The prepared photocurable cross-linked collagen gel was then quickly added. The adhesion of the gel to the wound surface was observed, and the wound surface was photographed. After 4 weeks of feeding, the wound site was observed. Figure 5 As shown in the photos (the leftmost is the modeling photo, the middle is the drug administration photo, and the rightmost is the photo 4 weeks later).

[0107] Depend on Figure 5 As can be seen in the photos, the gel is in a low-temperature liquid state (cures quickly at 32-37°C) and can be minimally injected through an arthroscopic approach, resulting in minimal surgical trauma and in line with the concept of minimally invasive surgery. The gel forms an ordered collagen fiber structure at the cartilage defect site, improving degradation resistance and mechanical strength. It integrates well with the surrounding cartilage, inducing chondrocyte proliferation and type II collagen secretion, achieving functional regeneration. After 4 weeks, the wound site was covered with new cartilage tissue, with no rejection reaction or inflammation, verifying the biocompatibility and repair efficacy of the material.

[0108] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage, characterized in that: The following steps are involved: (1) Weigh an appropriate amount of recombinant human collagen solution, add a pH adjuster to adjust the pH to 7, add methacrylic anhydride for modification, let it stand at 4°C, then add a separating agent, and centrifuge to obtain the collagen precipitate; (2) adding lysine oxidase to the collagen precipitate obtained in step (1) under stirring, mixing and cross-linking at 25° C., then transferring to an ultra-low temperature refrigerator and freezing for 4 h, transferring the frozen sample to a vacuum freeze dryer, and vacuum freeze-drying for 48 h to obtain a collagen sponge; (3) preparing a sodium hyaluronate solution, adding methacrylic anhydride to the sodium hyaluronate solution under ice bath and stirring conditions to prepare a sodium hyaluronate thermosensitive gel, and adding the collagen sponge to mix evenly; (4) converting the modified collagen sodium hyaluronate thermosensitive gel solution obtained in step (3) into a viscous gel at 32-37°C.

2. The method for preparing a collagen chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (1), the concentration of the recombinant human collagen solution is 1-2.5 mg / ml.

3. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (1), the mass ratio of the recombinant human collagen solution to methacrylic anhydride is (7-20):

1.

4. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (1), the pH adjuster comprises sodium hydroxide, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

5. The method for preparing a collagen chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (1), the 4°C standing time is 12 to 24 hours; The cross-linking time at 25° C. in step (2) is 6 to 12 hours.

6. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (1), the separating agent comprises ethanol, propylene glycol or glycerol.

7. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (2), the mass ratio of the recombinant human collagen precipitate to lysine oxidase is (15-50):

1.

8. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: The molecular weight of the sodium hyaluronate in step (3) is 100,000 to 250,000 Da.

9. The method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to claim 1, characterized in that: In step (3), the concentration of the sodium hyaluronate solution is 1-4 mg / ml; the mass ratio of the sodium hyaluronate solution to methacrylic anhydride is (18-25):

1.

10. A collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage prepared by the method for preparing a collagen-chitosan cross-linked thermosensitive gel for repairing articular cartilage according to any one of claims 1 to 9.

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