Biological crosslinking and chemical crosslinking combined collagen and preparation method thereof

Abstract

The invention discloses biological crosslinking and chemical crosslinking combined collagen and a preparation method thereof, and belongs to the technical field of medical hydrogel materials, the collagen is recombinant I-type collagen, and is obtained by fully mixing the recombinant I-type collagen with EDC / NHS, then adding lysyl oxidase and retreating, and the biological crosslinking and chemical crosslinking combined collagen is prepared. And swelling the recombinant type I collagen in normal saline to obtain the recombinant type I collagen hydrogel. The collagen and the recombinant I-type collagen hydrogel have the advantages of being low in cytotoxicity, excellent in cytocompatibility, not prone to causing collagen denaturation and the like.

Description

Technical Field

[0001] The present invention relates to a collagen combining biological crosslinking and chemical crosslinking and a preparation method thereof, belonging to the technical field of medical hydrogel materials. Background Art

[0002] Collagen is the main extracellular matrix molecule, which can self-assemble into fibrils with cross stripes by itself, provide support for cell growth, and be responsible for the mechanical elasticity of connective tissues. Collagen extracted at low temperature can still maintain the unique triple helix structure of collagen, and the prepared materials also have good flexibility, low immunogenicity, biocompatibility and biodegradability. However, natural collagen itself also has many defects, such as poor mechanical properties, poor water solubility resistance, and it is difficult to meet the use requirements without modification treatment. Therefore, it is usually necessary to modify the prepared collagen materials to improve their actual use performance.

[0003] Physical crosslinking and chemical crosslinking are the main crosslinking modification methods for collagen. The physical crosslinking of collagen is mainly carried out by methods such as ultraviolet irradiation, severe dehydration and heat treatment to generate crosslinks between collagen molecules, thereby improving the physical properties of collagen. The physical crosslinking method is likely to cause a certain degree of damage to collagen, resulting in collagen denaturation or degradation. Therefore, the physical crosslinking method is not very suitable for crosslinked collagen used in the manufacture of medical materials. Among chemical crosslinking methods, the most common is the use of glutaraldehyde. However, they have the risk of cytotoxicity and cause inflammatory reactions or tissue calcification, and there are certain limitations in applications in food, cosmetics or healthcare. Summary of the Invention

[0004] At least aiming at one of the above problems existing in the prior art, the present invention provides a collagen combining biological crosslinking and chemical crosslinking and a preparation method thereof. The collagen is obtained by fully mixing recombinant type I collagen with EDC / NHS and then adding lysyl oxidase for further treatment, that is, by a method combining biological crosslinking and chemical crosslinking; the recombinant type I collagen swells in physiological saline to form a recombinant type I collagen hydrogel, which has the advantages of low cytotoxicity, excellent cell compatibility and not easily causing collagen denaturation, etc.

[0005] To achieve the above object, the present invention adopts the following technical scheme: A collagen combining biological crosslinking and chemical crosslinking, the collagen is recombinant type I collagen, which is obtained by fully mixing recombinant type I collagen with EDC / NHS and then adding lysyl oxidase for further treatment.

[0006] Preferably, the recombinant type I collagen swells in physiological saline to obtain a recombinant type I collagen hydrogel, and the content of recombinant type I collagen is 25 - 45 mg / ml. If the content is too low, no gel can be formed; if the content is too high, it will become hard.

[0007] Preferably, in the recombinant type I collagen hydrogel, the content of recombinant type I collagen is 35 mg / ml.

[0008] The present invention also provides a method for preparing collagen by combining biological crosslinking and chemical crosslinking, and the specific steps are as follows: (1) Under stirring conditions, slowly dissolve EDC in phosphate buffer to make the final concentration of EDC 1.0 g / ml, adjust the pH of the solution to 4.5 - 5 to form an EDC solution, continue stirring, and add the freeze-dried recombinant type I collagen raw material. EDC activates the carboxylic acid part of aspartic acid and glutamic acid residues on the recombinant collagen, and an acylisourea group can be formed. Mix well to form mixed solution I; (2) Under the same stirring conditions, add NHS to mixed solution I. NHS converts the acylisourea group into an NHS-activated carboxylic acid group, which reacts with the amino group, and mix evenly to form mixed solution II; (3) Under the same stirring conditions, add lysyl oxidase to mixed solution II for biological crosslinking, and stir evenly to obtain a gel-like solid; (4) Freeze-dry the gel-like solid, crush it into particles with a diameter of 0.2 - 1.0 mm after freeze-drying, and then wash it with physiological saline to remove the residual crosslinking reagent in the gel to obtain recombinant type I collagen particles.

[0009] Preferably, in step (1), the mass ratio of the freeze-dried recombinant type I collagen raw material to EDC is 0.5 - 2:1, preferably 1:1.

[0010] Preferably, in step (2), the molar ratio of EDC to NHS is 1 - 4:1, preferably 4:1.

[0011] Preferably, in step (3), the mass ratio of the freeze-dried recombinant type I collagen raw material to lysyl oxidase is 10:0.5 - 2, preferably 10:1.

[0012] Preferably, it further includes step (5), swelling the washed recombinant type I collagen particles in physiological saline to obtain a recombinant type I collagen hydrogel, controlling the content of recombinant type I collagen to be 30 - 40 mg / ml, and filling it into a syringe.

[0013] Preferably, the stirring conditions in steps (1), (2) and (3) are: temperature 20 - 25°C, rotation speed 280 - 320 r / min.

[0014] Preferably, the sufficient mixing time in step (1) is 40 - 80 min; the uniform mixing time in step (2) is 40 - 80 min; the uniform stirring time in step (2) is 40 - 80 min; Preferably, the concentration of the phosphate buffer solution in step (1) is 250 - 350 mmol / L, preferably 300 mmol / L.

[0015] Preferably, the freeze-drying conditions in step (4) are: temperature 0 - 5°C, time 14 - 20 h; preferably temperature 4°C, time 16 h.

[0016] Preferably, the cleaning conditions in step (4) are: cleaning 4 - 5 times, each time for 25 - 35 min; the volume of normal saline used each time is 50 - 1200 times the weight of the gel-like solid.

[0017] The beneficial effects of the present invention: The collagen of the present invention is obtained by the method of combining biological crosslinking and chemical crosslinking, in which recombinant type I collagen is fully mixed by EDC / NHS and then further treated with lysyl oxidase. The recombinant type I collagen hydrogel formed by swelling the recombinant type I collagen in normal saline has the advantages of low cytotoxicity, excellent cell compatibility and not easily causing collagen denaturation, etc.; the recombinant type I collagen hydrogel of the present invention will not show the phenomenon of uneven dispersion or aggregation and concentration, which is beneficial for injection use; the recombinant type I collagen hydrogel of the present invention can not only accelerate wound hemostasis, but also accelerate the growth of wound fibroblasts and endothelial cells to promote wound healing, and at the same time effectively prevent wound exudate from adhering to the wound, avoid mechanical damage to the new granulation tissue again, and relieve the pain of the patient. After the wound heals, there is no scabbing or scarring phenomenon, and the skin is smooth; the recombinant type I collagen hydrogel of the present invention belongs to a self-repairing material and can be applied to fields such as implants, artificial skin, hemostatic sponges, scaffolds, medical devices, etc., and has broad application prospects. Description of the Drawings

[0018] Figure 1 It is the external view of the syringe filled with the recombinant type I collagen hydrogel of Example 1 and Comparative Examples 1 - 6 of the present invention; Figure 2 It is the push force curve graph of the recombinant type I collagen hydrogel of Example 1 of the present invention; Figure 3 It is the push force curve graph of the recombinant type I collagen hydrogel of Comparative Examples 1 to 6 of the present invention; Figure 4 It is the mechanical property graph of the recombinant type I collagen hydrogel of Example 1 and Example 4 of the present invention; Figure 5Comparison chart of the mechanical properties of the recombinant type I collagen hydrogels of Example 1, Example 4, Comparative Example 1, Comparative Example 3 and Comparative Example 4 of the present invention; Figure 6 Process of the recombinant type I collagen hydrogel of Example 1 of the present invention acting on a wound and recovery photos after 4 weeks; Figure 7 Recovery photos after 4 weeks of the recombinant type I collagen hydrogels of Comparative Examples 1 to 6 of the present invention acting on a wound. Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. For those conditions not specified in the embodiments, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents, instruments or components not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0020] Example 1 A preparation method of collagen and collagen hydrogel combining biological crosslinking and chemical crosslinking, the specific steps are as follows: (1) Under the stirring conditions of a temperature of 25°C and a rotation speed of 300 r / min, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was slowly dissolved in 10 ml of 300 mmol / L phosphate buffer solution to make the final concentration of EDC 1.0 g / ml. The pH of the solution was adjusted to 4.7 to form an EDC solution. Stirring was continued, and 10 g of freeze-dried recombinant type I collagen raw material was added. EDC activates the carboxylic acid parts of aspartic acid and glutamic acid residues on the recombinant collagen, and an acylisourea group can be formed. After thorough mixing for 60 min, a mixed solution I was formed; (2) Under the same stirring conditions, 1.498 g of N-hydroxysuccinimide (NHS) was added to the mixed solution I. NHS converts the acylisourea group into an NHS-activated carboxylic acid group, which reacts with the amino group. After thorough mixing for 60 min, a mixed solution II was formed; (3) Under the same stirring conditions, 1 g of lysyl oxidase was added to the mixed solution II for biological crosslinking, and stirring was carried out for 60 min to make it uniform, and a gel-like solid was obtained; (4) The gel-like solid was freeze-dried at 4 °C for 16 h, and after freeze-drying, it was crushed into particles with a diameter of 0.2 - 1.0 mm, and then placed in 50 - 1200 times the volume of normal saline and washed 5 times, 30 min each time, to remove the residual cross-linking reagent in the gel, obtaining recombinant type I collagen particles; (5) The washed recombinant type I collagen particles were swollen in normal saline to obtain a recombinant type I collagen hydrogel, and the content of recombinant type I collagen was controlled to be 35 mg / ml, and it was filled into a syringe, presenting a milky white gel state.

[0021] Example 2 A preparation method of collagen and collagen hydrogel combining biological cross-linking and chemical cross-linking, the specific steps are as follows: (1) Under the stirring condition of a temperature of 25 °C and a rotation speed of 320 r / min, EDC was slowly dissolved in 10 ml of 250 mmol / L phosphate buffer solution, so that the final concentration of EDC was 1.0 g / ml, the pH of the solution was adjusted to 4.5 to form an EDC solution, and stirring was continued. 5 g of freeze-dried recombinant type I collagen was added and mixed thoroughly for 40 min to form a mixed solution I; Among them, the mass ratio of the freeze-dried recombinant type I collagen raw material to EDC is 0.5:1; (2) Under the same stirring condition, 5.989 g of NHS was added to the mixed solution I and mixed thoroughly for 40 min to form a mixed solution II; (3) Under the same stirring condition, 1 g of lysyl oxidase was added to the mixed solution II for biological cross-linking and stirred for 40 min to be uniform, obtaining a gel-like solid; (4) The gel-like solid was freeze-dried at 0 °C for 14 h, and after freeze-drying, it was crushed into particles with a diameter of 0.2 - 1.0 mm, and then placed in 50 - 1200 times the volume of normal saline and washed 4 times, 25 min each time, to remove the residual cross-linking reagent in the gel, obtaining recombinant type I collagen particles; (5) The washed recombinant type I collagen particles were swollen in normal saline to obtain a recombinant type I collagen hydrogel, and the content of recombinant type I collagen was controlled to be 25 mg / ml, and it was filled into a syringe, presenting a milky white gel state.

[0022] Example 3 A preparation method of collagen and collagen hydrogel combining biological cross-linking and chemical cross-linking, the specific steps are as follows: (1) Under the stirring condition of a temperature of 20°C and a rotation speed of 300 r / min, slowly dissolve EDC in 10 ml of 350 mmol / L phosphate buffer solution so that the final concentration of EDC is 1.0 g / ml. Adjust the pH of the solution to 4.8 to form an EDC solution. Continue stirring, add 20 g of freeze-dried recombinant type I collagen, and mix well for 80 min to form mixed solution I; (2) Under the same stirring condition, add 2.995 g of NHS to mixed solution I, and mix well for 80 min to form mixed solution II; (3) Under the same stirring condition, add 1 g of lysyl oxidase to mixed solution II for biological crosslinking, and stir for 80 min until uniform to obtain a gel-like solid; (4) Freeze-dry the gel-like solid at a temperature of 5°C for 20 h. After freeze-drying, crush it into particles with a diameter of 0.2 - 1.0 mm, and then place it in 50 - 1200 times the volume of physiological saline and wash it 5 times, 35 min each time, to remove the residual crosslinking reagent in the gel and obtain recombinant type I collagen particles; (5) Swell the washed recombinant type I collagen particles in physiological saline to obtain a recombinant type I collagen hydrogel, and control the content of recombinant type I collagen to be 45 mg / ml. Fill it into a syringe, showing a milky white gel-like state.

[0023] Example 4 A preparation method of collagen and collagen hydrogel combining biological crosslinking and chemical crosslinking, the specific steps are as follows: (1) Under the stirring condition of a temperature of 20°C and a rotation speed of 300 r / min, slowly dissolve EDC in 10 ml of 300 mmol / L phosphate buffer solution so that the final concentration of EDC is 1.0 g / ml. Adjust the pH of the solution to 4.8 to form an EDC solution. Continue stirring, add 10 g of freeze-dried recombinant type I collagen, and mix well for 60 min to form mixed solution I; (2) Under the same stirring condition, add 1.99 g of NHS to mixed solution I, and mix well for 50 min to form mixed solution II; (3) Under the same stirring condition, add 0.5 g of lysyl oxidase to mixed solution II for biological crosslinking, and stir for 60 min until uniform to obtain a gel-like solid; (4) Freeze-dry the gel-like solid at a temperature of 4°C for 18 h. After freeze-drying, crush it into particles with a diameter of 0.2 - 1.0 mm, and then place it in 50 - 1200 times the volume of physiological saline and wash it 4 times, 30 min each time, to remove the residual crosslinking reagent in the gel and obtain recombinant type I collagen particles; (5) Swell the washed recombinant type I collagen particles in physiological saline to obtain a recombinant type I collagen hydrogel, control the content of recombinant type I collagen to be 30 mg / ml, and fill it into a syringe, presenting a milky white gel-like state.

[0024] Comparative Example 1 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: step (3) is not carried out. After the complete physical cross-linking is completed in step (2), the liquid is removed to obtain a gel-like solid, and step (4) is directly carried out; the rest is exactly the same.

[0025] Comparative Example 2 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: (1) Under the stirring conditions of a temperature of 25 °C and a rotation speed of 300 r / min, add the freeze-dried recombinant type I collagen to the 300 mmol / L phosphate buffer solution with the pH adjusted to 4.7, and mix evenly to form a mixed solution I; step (2) is not carried out, and step (3) is directly carried out: add lysyl oxidase to the mixed solution I for biological cross-linking treatment; without the physical cross-linking treatment of EDC / NHS; the rest is exactly the same.

[0026] Comparative Example 3 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: step (2) is not included, and step (3) is directly carried out: add lysyl oxidase to the mixed solution I for biological cross-linking treatment; only the combination of EDC physical cross-linking and biological cross-linking; the rest is exactly the same.

[0027] Comparative Example 4 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: (1) Under the stirring conditions of a temperature of 25 °C and a rotation speed of 300 r / min, slowly dissolve NHS in the 300 mmol / L phosphate buffer solution to make the final concentration of NHS 0.1853 g / ml, adjust the pH of the solution to 4.7 to form an NHS solution, continue stirring, add the freeze-dried recombinant type I collagen, and mix well to form a mixed solution I; step (3) is directly carried out: add lysyl oxidase to the mixed solution I for biological cross-linking treatment; only the combination of NHS physical cross-linking and biological cross-linking; the rest is exactly the same.

[0028] Comparative Example 5 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: (1) Under the stirring condition of a temperature of 25 °C and a rotation speed of 300 r / min, NHS was slowly dissolved in 300 mmol / L phosphate buffer solution to make the final concentration of NHS 0.1853 g / ml. The pH of the solution was adjusted to 4.7 to form an NHS solution. Stirring was continued, and freeze-dried recombinant type I collagen was added and mixed thoroughly to form mixed solution I; (2) Under the same stirring condition, EDC was added to mixed solution I and mixed evenly to form mixed solution II; wherein, the molar ratio of EDC to NHS was 4:1; the rest was exactly the same.

[0029] Comparative Example 6 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: the freeze-dried recombinant type I collagen used as a raw material was swollen in physiological saline to obtain a recombinant type I collagen hydrogel with a recombinant collagen content of 35 mg / ml, and then filled into a syringe.

[0030] Comparative Example 7 A preparation method of collagen and collagen hydrogel, which is different from Example 1 in that: (1) Under the stirring condition of a temperature of 25 °C and a rotation speed of 300 r / min, freeze-dried recombinant type I collagen was added to 300 mmol / L phosphate buffer solution with the pH adjusted to 4.7 and mixed evenly to form mixed solution I; Step (2) was not carried out, and step (3) was directly carried out: glutaraldehyde was added to mixed solution I for biocrosslinking treatment; the physical crosslinking treatment of EDC / NHS was not included; the rest was exactly the same.

[0031] The freeze-dried recombinant type I collagen raw materials used in the above Examples 1 to 4 and Comparative Examples 1 to 7 were prepared according to CN118995817A.

[0032] Effect Test Example The recombinant type I collagen hydrogels of the above Example 1 and Comparative Examples 1 to 7 were characterized as follows: 1. Appearance The recombinant type I collagen hydrogels of Example 1 and Comparative Examples 1 to 7 were filled into syringes and the appearance state was observed.

[0033] From Figure 1It can be seen from the figure that after being filled in the syringe, the recombinant type I collagen hydrogel of Example 1 presents a uniform milky white gel, which is clear, free of impurities and has a smooth surface, while the gel state of the recombinant type I collagen hydrogels of Comparative Examples 1 to 7 is poor. The recombinant type I collagen hydrogel of Comparative Example 1 presents a colorless and transparent gel with bubbles. The recombinant type I collagen hydrogel of Comparative Example 2 is uneven in color, with milky white, colorless and transparent mixed, unevenly dispersed, and bubbles. The recombinant type I collagen hydrogel of Comparative Example 3, Comparative Example 4 and Comparative Example 5 is uneven in color, with milky white, colorless and transparent mixed, unevenly dispersed, and bubbles. Although the type I collagen hydrogels in group I also present a milky white gel, there are certain bubbles. The recombinant type I collagen hydrogel in comparative example 6 presents a transparent gel with low transparency, aggregation, rough surface, and bubbles. The recombinant type I collagen hydrogel in comparative example 7 presents a transparent gel, which is turbid and has bubbles. Therefore, the gel state of the recombinant type I collagen hydrogel prepared by the present invention is better, which is a milky white gel, uniform, clear, and free of impurities. It can maintain a stable structure in the syringe and is not easy to deform or break.

[0034] 2. Syringe pushing force The recombinant type I collagen hydrogels of Example 1 and Comparative Examples 1 to 6 were filled into a syringe, and the syringe push rod was pushed at a constant speed. During the experiment, a 27G injection needle was installed to simulate actual use. The push rod was pushed at a constant speed of 30 mm / min, and the sample in the syringe was pushed out through the needle; and the pushing force curve was obtained.

[0035] from Figure 2 and Figure 3 In the figure, we can see the change of pushing force during the sample extrusion process. When the pushing force is small, the sample is easy to be extruded, and when the pushing force is large, the sample is not easy to be extruded.

[0036] pass Figure 2 The recombinant type I collagen hydrogel in Example 1 exhibits an appropriate pushing force during the injection process, which is neither too large nor too small, so that the injection process is smooth and controllable, the injection efficiency is high, and it can maintain a complete gel state and can diffuse evenly in the skin, which is conducive to good integration with human tissues and promote tissue regeneration and repair.

[0037] pass Figure 3 The sample in comparative example 2 is unevenly dispersed, and the sample in comparative example 6 is aggregated and concentrated, resulting in a large difference in the height of the pushing force. In comparative example 5, accumulation occurs during the pushing process, and the pushing force increases. In this way, the pushing force may be too large or too small, resulting in difficult and uncontrollable injection, uneven diffusion, affecting the injection effect and safety, and also affecting the hand suitability during injection.

[0038] 3. Mechanical properties The recombinant type I collagen hydrogels of Example 1, Example 4, Comparative Example 1, Comparative Example 3, and Comparative Example 4 were subjected to frequency scanning at a shear rate of 0.01 Hz - 100 Hz using a rheometer at a temperature of 25 ± 2°C, and a coordinate graph was plotted with the log values of the viscous modulus and elastic modulus against the log values of the frequency.

[0039] As Figure 4 shown, for the recombinant type I collagen hydrogel sample 3 - 2 of Example 1 and the recombinant type I collagen hydrogel sample 3 - 1 of Example 4, the storage modulus is much greater than the loss modulus, indicating high elasticity and low viscosity. With the change of frequency, it maintains good elasticity, and as the frequency increases, its elasticity enhances, and the material property stability is better.

[0040] In addition, combining Figure 5 with the graph of storage modulus - angular frequency, comparing the recombinant type I collagen hydrogel sample 3 - 2 of Example 1 in A with the recombinant type I collagen hydrogel sample 1 - 1 of Comparative Example 1 and the recombinant type I collagen hydrogel sample 2 - 1 of Comparative Example 3, the storage modulus of the recombinant type I collagen hydrogel of Example 1 is much greater than those of Comparative Example 1 and Comparative Example 3, indicating that the recombinant type I collagen hydrogels of Comparative Example 1 and Comparative Example 3 have low elasticity; comparing the recombinant type I collagen hydrogel sample 3 - 1 of Example 1 in B with the recombinant type I collagen hydrogel sample 1 - 2 of Comparative Example 1 and the recombinant type I collagen hydrogel sample 2 - 2 of Comparative Example 4, the storage modulus of the recombinant type I collagen hydrogel of Example 4 is much greater than those of Comparative Example 1 and Comparative Example 4, indicating that the recombinant type I collagen hydrogels of Comparative Example 1 and Comparative Example 4 have low elasticity, further demonstrating that the recombinant type I collagen hydrogel prepared by the present invention has high elasticity.

[0041] 4. Stability The recombinant type I collagen hydrogels of Examples 1 - 4 and Comparative Examples 1 - 6 were evaluated for thermal stability through appearance, syringe extrusion force, and mechanical properties. The gel changes of the recombinant type I collagen hydrogels of Examples 1 - 4 and Comparative Examples 1 - 6 before aging at 2 - 4°C and 25°C and after accelerated aging at 25°C for 72 days are shown in the stability in Table 1 below.

[0042] Table 1 Stability of recombinant type I collagen hydrogel

[0043] As can be seen from Table 1, the recombinant type I collagen of the present invention is first mixed with EDC and then with NHS, and then cross-linked with lysyl oxidase, sheared to form particles, washed, and swollen to obtain the recombinant type I collagen hydrogel. It is milky white gel-like, clear and impurity-free, and there is no obvious difference in accelerated aging at 2-4°C, before aging at 25°C, and after 72 days of aging at 25°C, showing good stability.

[0044] In addition, through the tests of syringe pushing force, mechanical properties and stability, it is found that the recombinant type I collagen hydrogel of the present invention has a moderate cross-linking degree, a concentration of 25-45 mg / ml, strong elasticity and more appropriate viscosity. That is, it maintains a complete gel state and makes the injection smooth and controllable, and the material properties have good stability.

[0045] 5. Cytotoxicity experiment 5.1 Sample preparation Extract the samples according to the ratio in Table 2 below (sample: extract volume), and extract them in a constant temperature shaking incubator at 37°C and 60 rpm for 24 hours; after the extraction, check the extraction changes, and the extract is immediately used for the experiment without filtration, centrifugation, dilution, etc., and the pH is not adjusted; at the same time, prepare blank control, negative control and positive control samples.

[0046] Table 2 Sample preparation conditions

[0047] 5.2 Test method preparation The experiment is carried out under aseptic conditions: culture L-929 cells in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin, streptomycin), and place them in an incubator at 37°C and 5% CO2 for culture; digest the cells with trypsin to make a cell suspension, centrifuge (200G, 3 min), and then redisperse the cells in fresh medium to adjust the cell density to 1×10 5Cell suspension at cells / ml; Inoculate the above cell suspension into a 96-well culture plate, 100 μl per well, and culture in a carbon dioxide incubator for 24 hours (5% CO2, temperature 37 °C, humidity > 90%); After the cells grow into a monolayer, aspirate the original culture medium, and add 100 μl of test samples at different concentrations (100%, 75%, 50%, 25%), blank control solution, positive control solution (100%), and negative control solution (100%) respectively, and culture at 37 °C and 5% CO2 for 24 hours. Each group has 6 parallel samples; After culturing for 24 hours, take out the 96-well plate, observe cell morphology under a microscope, then aspirate the liquid, add 50 μl of MTT (1 mg / ml) to each well, culture in a carbon dioxide incubator for 2 hours, discard the MTT solution, and add 100 μl of isopropanol solution to each well; Shake the plate and measure the absorbance at 570 nm on an enzyme-linked immunosorbent assay (ELISA) reader (reference wavelength 650 nm).

[0048] 5.3 After the extract acts on the cells, observe the morphology under the microscope, and the cell morphology is described in Table 3 below.

[0049] Table 3 Description of cell morphology

[0050] 5.4 Evaluation criteria 1) When the survival rate is relatively low, the potential cytotoxicity of the test sample is relatively high; 2) If the survival rate drops to less than 70% of the blank, it has potential cytotoxicity; 3) The survival rate of the 50% extract of the test sample should be at least the same as or higher than that of the 100% extract. Otherwise, the experiment should be repeated; 4) The survival rate of the 100% extract of the test sample is the final result.

[0051] Perform a cytotoxicity test on the recombinant type I collagen hydrogel of Example 1 and Comparative Example 7, and the results are shown in Table 4 below.

[0052] Table 4 Cytotoxicity test

[0053] It is found from the table that under the test conditions of this time, the recombinant type I collagen hydrogel of Example 1 has no potential toxic effect on L-929 cells; while the recombinant type I collagen hydrogel crosslinked with glutaraldehyde in Comparative Example 7 has no potential toxic effect on L-929 cells.

[0054] 6. Animal experiments Using rats as an animal model, they were anesthetized by injecting 2 ml of 2.5% sodium pentobarbital solution. Two 1 cm × 1 cm wounds were made on the back of the rats, and drugs were administered separately. During the formation of the wounds, the bleeding was absorbed with gauze, and then the prepared recombinant type I collagen hydrogel was quickly applied to the wounds. The complete hemostasis time was recorded, the adhesion of the material to the wounds was observed, and the wound surface was photographed; they were raised for 0 - 4 weeks and then the wound sites were observed; among them, for one rat, one wound was administered with the recombinant type I collagen hydrogel of Example 1, and one wound was not administered; the recombinant type I collagen hydrogels obtained in Comparative Examples 1 - 6 were combined in pairs and administered to the two wounds of one rat. The complete hemostasis time, the wounds and their surface conditions are as shown in Table 5, Figure 6 and 7 shown below.

[0055] Figure 6 Figure Figure 6 shows the process of the recombinant type I collagen hydrogel of Example 1 acting on the wound of one rat - drug administration - recovery and the condition of the wound site 4 weeks later, Figure 7 Figure 7 shows the condition of the wound site 4 weeks later when the recombinant type I collagen hydrogels obtained in Comparative Examples 1 - 6 act on the rats; Figure 6 The COL1A1 emulsion in Figure 6 is the recombinant type I collagen hydrogel of Example 1; Figure 7 COL1B1 to COL1B6 in Figure 7 respectively correspond to the recombinant type I collagen hydrogels of Comparative Examples 1 - 6 one by one.

[0056] Table 5 Wound Conditions

[0057] As shown in Table 5, Figure 6 and 7 shown below, when using the recombinant type I collagen hydrogel of Example 1, the wound hemostasis is accelerated, and the wound completely heals after 0.7 weeks of raising. After 4 weeks, the wound heals without scabbing or scarring, and the skin is smooth; while for the recombinant type I collagen hydrogels of Comparative Examples 1 - 6, the wound hemostasis time is long, the wound healing time is relatively slow, the wound healing has scabbing or scarring phenomena, and the wound without drug administration has scarring phenomena.

[0058] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above - mentioned exemplary embodiments, and without departing from the spirit and basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non - restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0059] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A collagen combined with biological crosslinking and chemical crosslinking, characterized in that The collagen is recombinant type I collagen, which is obtained by fully mixing recombinant type I collagen with EDC / NHS and then further treating with lysyl oxidase.

2. The collagen combined with biological crosslinking and chemical crosslinking according to claim 1, characterized in that the recombinant type I collagen is swollen in physiological saline to obtain a recombinant type I collagen hydrogel, and the content of recombinant type I collagen is 25-45 mg / ml.

3. The collagen combined with biological crosslinking and chemical crosslinking according to claim 2, characterized in that in the recombinant type I collagen hydrogel, the content of recombinant type I collagen is 35 mg / ml.

4. A method for preparing a collagen combined with biological crosslinking and chemical crosslinking according to claim 1, characterized in that, It includes the following steps: (1) Under stirring conditions, dissolve EDC in phosphate buffer solution to make the final concentration of EDC 1.0 g / ml, adjust the pH of the solution to 4.5-5 to form an EDC solution, continue stirring, and add freeze-dried recombinant type I collagen, and mix well to form a mixed solution I; (2) Under the same stirring conditions, add NHS to the mixed solution I and mix evenly to form a mixed solution II; (3) Under the same stirring conditions, add lysyl oxidase to the mixed solution II for biological crosslinking, and stir evenly to obtain a gel-like solid; (4) Freeze-dry the gel-like solid, crush it into particles with a diameter of 0.2-1.0 mm after freeze-drying, and then wash with physiological saline to remove the residual crosslinking reagent in the gel to obtain recombinant type I collagen particles.

5. The preparation method of the collagen combined with biological crosslinking and chemical crosslinking according to claim 4, characterized in that, The mass ratio of the freeze-dried recombinant type I collagen to EDC is 0.5-2:

1.

6. The preparation method of the collagen combining biological crosslinking and chemical crosslinking according to claim 4, characterized in that, The molar ratio of EDC to NHS is 1-4:

1.

7. The preparation method of the collagen combined with biological crosslinking and chemical crosslinking according to claim 4, wherein, The mass ratio of the freeze-dried recombinant type I collagen raw material to lysyl oxidase is 10:0.5-2.

8. The preparation method of the collagen combining biological crosslinking and chemical crosslinking according to claim 4, characterized in that, It further includes step (5), swelling the washed recombinant type I collagen particles in physiological saline to obtain a recombinant type I collagen hydrogel, controlling the content of recombinant type I collagen to be 25-45 mg / ml, and filling it into a syringe.

9. The preparation method of the collagen combined with biological crosslinking and chemical crosslinking according to claim 4, characterized in that, The stirring conditions for steps (1) to (3): temperature 20-25 °C, rotation speed 280-320 r / min; the concentration of the phosphate buffer solution in step (1) is 250-350 mmol / L.

10. The preparation method of the collagen combined with biological crosslinking and chemical crosslinking according to claim 4, characterized in that, For step (4), the freeze-drying conditions: temperature 0-5 °C, time 14-18 h; The washing conditions: wash 4-5 times, each time for 25-35 min; the volume of physiological saline used each time is 50-1200 times the weight of the gel-like solid.

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