Medical recombinant collagen repair gel and preparation method thereof

Abstract

The present application relates to a kind of medical recombinant collagen repair gel and its preparation method, belong to the technical field of recombinant collagen.The present application improves the tissue repair effect significantly by the synergistic effect of collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drug and cell adhesion peptide.The multistage pore size distribution is generated by gas foaming method, staged freezing and freeze-drying, and the gel with porous structure is formed, which is beneficial to cell penetration and tissue regeneration;And the permeability and drug loading capacity of the gel are improved, and the cell adhesion peptide, growth factor and anti-inflammatory drug are better absorbed to the gel, functional sustained release is realized, and the repair effect is further improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of recombinant collagen, and relates to a medical recombinant collagen repair gel and a preparation method thereof. BACKGROUND

[0002] Collagen, as the main component of human extracellular matrix, plays an irreplaceable role in tissue repair and regeneration. In recent years, medical recombinant collagen repair gel has gradually become a research hotspot in the fields of wound repair, burn treatment and medical cosmetology due to its excellent biocompatibility, controllable degradability and low immunogenicity. Compared with traditional animal-derived collagen, recombinant collagen can accurately simulate the amino acid sequence of natural collagen by expressing specific functional domains through genetic engineering technology, significantly reducing the risk of immune rejection and avoiding viral contamination problems. Such gel can simulate the extracellular microenvironment through the three-dimensional network structure of the loaded recombinant collagen, promote cell adhesion, proliferation and migration, and accelerate wound healing. However, its clinical application still faces technical bottlenecks such as insufficient mechanical properties of the gel, poor stability and low release efficiency of active ingredients, which need to be broken through by optimizing the preparation process and functional design.

[0003] In the prior art, the preparation of collagen gel is mostly based on collagen extracted from animals, but in the past, most of the collagen scaffolds were extracted from the tendons of cows and horses, and then reconstructed into collagen scaffolds using glutaraldehyde and other crosslinking agents. However, due to the immunogenicity risk, instability and batch inconsistency of collagen derived from natural tissues, in recent years, recombinant collagen technology has developed rapidly. Recombinant collagen is synthesized in microorganisms or other expression systems through genetic engineering means, which can avoid the immunogenicity problem of natural collagen and improve the controllability and purity of production. Therefore, the medical repair gel developed based on recombinant collagen not only has the advantages of natural collagen, but also has more superior physical and chemical properties and biological safety, becoming a research hotspot in the field of tissue repair.

[0004] The collagen extracted from animal sources is not only expensive, but also destroys the natural structure between collagen bundles, and it is difficult to meet the mechanical and biological performance requirements of bone replacement materials. At the same time, it may have high resistance to tissue. With the development of genetic engineering technology, the preparation of recombinant collagen has gradually become the mainstream, such as using Pichia pastoris expression system to produce recombinant human type III collagen, and forming a gel through ion cross-linking method, but the gel network is loose, and it is difficult to maintain long-term structural stability. In addition, the blending modification strategy is often used in the prior art to compound the recombinant collagen with hyaluronic acid, and the hydrogen bond action is used to enhance the water holding capacity of the gel, but the problem of local high concentration caused by burst release of active ingredients has not been solved. Some studies attempt to introduce photo-crosslinking technology, and realize the photo-curing forming of the gel through methacrylation modification, however, the photo initiator may cause cytotoxicity, and the complex modification process is easy to destroy the natural conformation of collagen. Therefore, developing a kind of efficient, safe and controllable medical recombinant collagen repair gel preparation method has become a key problem to be solved. SUMMARY

[0005] The application relates to a medical recombinant collagen repair gel and a preparation method thereof, and belongs to the technical field of recombinant collagen. The medical recombinant collagen repair gel is prepared by the synergistic action of collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, an anti-inflammatory drug and a cell adhesion peptide, so that the tissue repair effect is significantly improved. The gas foaming method, the staged freezing and the freeze drying are used to generate a multi-level pore size distribution, so that the gel with a porous structure is formed, cell penetration and tissue regeneration are facilitated, the permeability and the drug loading capacity of the gel are improved, the cell adhesion peptide, the growth factor and the anti-inflammatory drug are better adsorbed on the gel, functional sustained release is realized, and the repair effect is further improved.

[0006] The object of the application can be achieved by the following technical scheme.

[0007] The medical recombinant collagen repair gel comprises the following raw materials in parts by weight: 40-50 parts of collagen, 15-20 parts of chitosan, 10-15 parts of polylactic acid, 5-10 parts of nanocellulose, 1-2 parts of vascular endothelial growth factor, 1-3 parts of an anti-inflammatory drug and 1-2 parts of a cell adhesion peptide.

[0008] As a preferred technical scheme of the application, the anti-inflammatory drug is any one of dexamethasone, indomethacin and ibuprofen; and the cell adhesion peptide is any one of RGD peptide and fibronectin.

[0009] Further, the preparation method of the medical recombinant collagen repair gel comprises the following steps.

[0010] (1) collagen is dissolved in 0.1M glacial acetic acid solution, stirred until completely dissolved to obtain a collagen solution, then chitosan is dissolved in 0.1M glacial acetic acid solution, stirred until transparent to obtain a chitosan solution, the collagen solution is added dropwise into the chitosan solution, stirred uniformly to form a mixed solution, polylactic acid is dispersed in anhydrous ethanol and ultrasonically treated for 10-30min to obtain a polylactic acid solution, the polylactic acid solution is added dropwise into the mixed solution, and stirring is uniformly performed to prepare a matrix solution;

[0011] (2) nanocellulose is dispersed in deionized water, ultrasonically treated for 10-20min to mix uniformly to form a suspension, the suspension is added dropwise into the matrix solution, stirring is uniformly performed, 1M sodium hydroxide solution is used to adjust the pH to 6.5-7.4, a crosslinking agent is added, and stirring is uniformly performed to prepare a basic gel solution;

[0012] (3) the basic gel solution is placed in a sealed container, a foaming gas is injected through a micro gas injection device, the crosslinking agent is added to a concentration of 10-20mmol / L, and stirring is performed at 4℃ for 12 hours, -10~-100kPa vacuum treatment is performed for 30min, and residual gas is removed;

[0013] (4) the gel solution foamed by the gas is frozen at a temperature of-80℃ for 1-2 hours, and then frozen at-20℃ for 4-6 hours, and the basic gel is prepared through vacuum freeze drying;

[0014] (5) the crosslinking agent is added to a cell adhesion peptide solution with a concentration of 1mg / mL, stirring is performed for 20-40min, the basic gel is added, and soaking is performed for 3-5h, then the cell adhesion gel is prepared by removing the unbound cell adhesion peptide and crosslinking agent through PBS with pH 7.4;

[0015] (6) the cell adhesion gel is soaked in a solution containing 10-50ng / mL vascular endothelial growth factor and 10-100μg / mL anti-inflammatory drug, the growth factor and the anti-inflammatory drug are allowed to enter the pores of the gel through diffusion adsorption, the unbound growth factor is removed through washing with PBS with pH 7.4, and vacuum drying is performed at 10-40℃ for 2-4h;

[0016] (7) 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide is added to a concentration of 5-15mmol / L and N-hydroxysuccinimide is added to a concentration of 2-8mmol / L, and crosslinking is performed by stirring at 1-9℃ for 10-14h to obtain the medical recombinant collagen repair gel.

[0017] As a preferred technical scheme of the present application, the collagen solution in step (1) has a concentration of 1-2wt%; the chitosan solution has a concentration of 1-2wt%; and the polylactic acid solution has a concentration of 5-10wt%.

[0018] As a preferred technical solution of the present invention, the crosslinking agent in step (2) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution and crosslinking agent is 1-5:70-85:10-20:0.5-1.

[0019] As a preferred technical solution of the present invention, the crosslinking agent in step (3) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the foaming gas is carbon dioxide or nitrogen; the injection pressure of the foaming gas is 50-200 kPa and the injection time is 5-15 min.

[0020] As a preferred technical solution of the present invention, the vacuum freeze-drying conditions in step (4) are -50°C, vacuum degree is 5-15Pa, and vacuum freeze-drying time is 24-48h.

[0021] As a preferred embodiment of the present invention, the crosslinking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or N-hydroxysuccinimide; the mass ratio of the cell adhesion peptide solution, crosslinking agent, base gel and PBS is 1:0.01-0.2:5-15:15-25.

[0022] As a preferred technical solution of the present invention, the mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS in step (6) is 1:3-5:8-12.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention combines various biomaterials and active molecules, such as collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drugs, and cell adhesion peptides, to endow the gel with multifunctionality. Collagen provides good biocompatibility and cell adhesion ability, chitosan has antibacterial and wound-healing properties, and polylactic acid provides mechanical strength and controllable degradation performance. The introduction of nanocellulose further enhances the mechanical strength and structural stability of the gel, while forming a three-dimensional support framework, providing an ideal microenvironment for cell adhesion and proliferation. This multi-component composite design not only significantly improves the bioactivity and functionality of the gel, but also allows for adaptation to different tissue repair needs by adjusting the proportion of each component.

[0025] (2) This invention employs carbon dioxide or nitrogen for foaming treatment, resulting in a more uniform pore distribution and significantly improving the gel's permeability and drug loading capacity, thereby enabling the controlled release of growth factors and anti-inflammatory drugs. Simultaneously, staged freezing creates a three-dimensional porous structure with gradient pore sizes, enhancing the gel's cell permeability and tissue regeneration capabilities. Furthermore, the cross-linking technology using 1-ethyl-(3-dimethylaminopropyl)carboimide and N-hydroxysuccinimide effectively cross-links collagen and chitosan, ensuring the gel's mechanical properties remain stable while maintaining its biocompatibility. Detailed Implementation

[0026] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0027] Example 1

[0028] A medical recombinant collagen repair gel comprises the following raw materials in parts by weight: 45 parts collagen, 18 parts chitosan, 13 parts polylactic acid, 7 parts nanocellulose, 1.5 parts vascular endothelial growth factor, 2 parts anti-inflammatory drugs, and 1.5 parts cell adhesion peptides.

[0029] The anti-inflammatory drug is dexamethasone; the cell adhesion peptide is RGD peptide.

[0030] The preparation method of the aforementioned medical recombinant collagen repair gel includes the following steps:

[0031] (1) Dissolve collagen in 0.1M glacial acetic acid solution and stir until completely dissolved to obtain collagen solution. Then dissolve chitosan in 0.1M glacial acetic acid solution and stir until transparent to obtain chitosan solution. Add collagen solution dropwise to chitosan solution and stir evenly to obtain mixed solution. Disperse polylactic acid in anhydrous ethanol and sonicate for 20 min to obtain polylactic acid solution. Add polylactic acid solution dropwise to mixed solution and stir evenly to obtain matrix solution.

[0032] (2) Disperse nanocellulose in deionized water, sonicate for 15 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 7.0 with 1M sodium hydroxide solution, add crosslinking agent and stir evenly to obtain the basic gel solution;

[0033] (3) Place the base gel solution in a sealed container, inject foaming gas through a micro gas injection device, add crosslinking agent to a concentration of 15 mmol / L, stir at 4°C for 12 hours, and treat under -50 kPa vacuum for 30 min to remove residual gas.

[0034] (4) The gas-foamed gel solution was frozen at -80°C for 1.5 hours and then frozen at -20°C for 5 hours. The base gel was obtained by vacuum freeze drying.

[0035] (5) Add cross-linking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 30 min, add basic gel, soak for 4 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptides and cross-linking agent to obtain cell adhesion gel.

[0036] (6) The cell adhesion gel was immersed in a solution containing 30 ng / mL vascular endothelial growth factor and 55 μg / mL anti-inflammatory drug. The growth factor and anti-inflammatory drug were allowed to enter the pores of the gel through diffusion adsorption. The gel was washed with PBS at pH 7.4 to remove unbound growth factor and then vacuum dried at 20°C for 3 h.

[0037] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 10 mmol / L and N-hydroxysuccinimide to a concentration of 5 mmol / L, and stir at 4°C for 12 h to crosslink the mixture, thereby obtaining medical recombinant collagen repair gel.

[0038] The collagen solution concentration in step (1) is 1.5 wt%; the chitosan solution concentration is 1.5 wt%; and the polylactic acid solution concentration is 8 wt%.

[0039] The crosslinking agent in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the nanocellulose, deionized water, matrix solution and crosslinking agent is 3:80:15:0.8.

[0040] The crosslinking agent in step (3) is N-hydroxysuccinimide; the foaming gas is carbon dioxide; the injection pressure of the foaming gas is 150 kPa and the injection time is 10 min.

[0041] The vacuum freeze-drying conditions in step (4) are -50℃, vacuum degree of 10Pa, and vacuum freeze-drying time of 36h.

[0042] The cross-linking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, cross-linking agent, base gel and PBS is 1:0.1:10:20.

[0043] In step (6), the mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS is 1:4:10.

[0044] Example 2

[0045] A medical recombinant collagen repair gel comprises the following raw materials in parts by weight: 40 parts collagen, 15 parts chitosan, 10 parts polylactic acid, 5 parts nanocellulose, 1 part vascular endothelial growth factor, 1 part anti-inflammatory drug, and 1 part cell adhesion peptide.

[0046] The anti-inflammatory drug is indomethacin; the cell adhesion peptide is fibronectin.

[0047] The preparation method of the aforementioned medical recombinant collagen repair gel includes the following steps:

[0048] (1) Dissolve collagen in 0.1M glacial acetic acid solution and stir until completely dissolved to obtain collagen solution. Then dissolve chitosan in 0.1M glacial acetic acid solution and stir until transparent to obtain chitosan solution. Add collagen solution dropwise to chitosan solution and stir evenly to obtain mixed solution. Disperse polylactic acid in anhydrous ethanol and sonicate for 10 min to obtain polylactic acid solution. Add polylactic acid solution dropwise to mixed solution and stir evenly to obtain matrix solution.

[0049] (2) Disperse nanocellulose in deionized water, sonicate for 10 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 6.5 with 1M sodium hydroxide solution, add crosslinking agent and stir evenly to obtain the basic gel solution;

[0050] (3) Place the base gel solution in a sealed container, inject foaming gas through a micro gas injection device, add crosslinking agent to a concentration of 10 mmol / L, stir at 4°C for 12 hours, and vacuum-treat at -10 kPa for 30 min to remove residual gas.

[0051] (4) The gas-foamed gel solution was frozen at -80°C for 1 hour, and then frozen at -20°C for 4 hours. The base gel was obtained by vacuum freeze drying.

[0052] (5) Add cross-linking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 20 min, add basic gel, soak for 3 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptides and cross-linking agent to obtain cell adhesion gel.

[0053] (6) The cell adhesion gel was immersed in a solution containing 10 ng / mL vascular endothelial growth factor and 10 μg / mL anti-inflammatory drug. The growth factor and anti-inflammatory drug were allowed to enter the pores of the gel through diffusion adsorption. The gel was washed with PBS at pH 7.4 to remove unbound growth factor and then vacuum dried at 10°C for 2 h.

[0054] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 5 mmol / L and N-hydroxysuccinimide to a concentration of 2 mmol / L, and stir at 1°C for 10 h to crosslink the mixture, thereby obtaining medical recombinant collagen repair gel.

[0055] In step (1), the collagen solution concentration is 1-2 wt%; the chitosan solution concentration is 1 wt%; and the polylactic acid solution concentration is 5 wt%.

[0056] The crosslinking agent in step (2) is N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution and crosslinking agent is 1:70:10:0.5.

[0057] The crosslinking agent in step (3) is N-hydroxysuccinimide; the foaming gas is nitrogen; the injection pressure of the foaming gas is 50 kPa and the injection time is 15 min.

[0058] The vacuum freeze-drying conditions in step (4) are -50℃, vacuum degree is 15Pa, and vacuum freeze-drying time is 24h.

[0059] The cross-linking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, cross-linking agent, base gel and PBS is 1:0.01:5:15.

[0060] In step (6), the mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS is 1:3:8.

[0061] Example 3

[0062] A medical recombinant collagen repair gel comprises the following raw materials in parts by weight: 50 parts collagen, 20 parts chitosan, 15 parts polylactic acid, 10 parts nanocellulose, 2 parts vascular endothelial growth factor, 3 parts anti-inflammatory drugs, and 2 parts cell adhesion peptides.

[0063] The anti-inflammatory drug is ibuprofen; the cell adhesion peptide is RGD peptide.

[0064] The preparation method of the aforementioned medical recombinant collagen repair gel includes the following steps:

[0065] (1) Dissolve collagen in 0.1M glacial acetic acid solution and stir until completely dissolved to obtain collagen solution. Then dissolve chitosan in 0.1M glacial acetic acid solution and stir until transparent to obtain chitosan solution. Add collagen solution dropwise to chitosan solution and stir evenly to obtain mixed solution. Disperse polylactic acid in anhydrous ethanol and sonicate for 30 min to obtain polylactic acid solution. Add polylactic acid solution dropwise to mixed solution and stir evenly to obtain matrix solution.

[0066] (2) Disperse nanocellulose in deionized water, sonicate for 20 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 7.4 with 1M sodium hydroxide solution, add crosslinking agent and stir evenly to obtain the basic gel solution;

[0067] (3) Place the base gel solution in a sealed container, inject foaming gas through a micro gas injection device, add crosslinking agent to a concentration of 20 mmol / L, stir at 4°C for 12 hours, and treat under -100 kPa vacuum for 30 min to remove residual gas.

[0068] (4) The gas-foamed gel solution was frozen at -80°C for 2 hours, and then frozen at -20°C for 6 hours. The base gel was obtained by vacuum freeze drying.

[0069] (5) Add cross-linking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 40 min, add basic gel, soak for 5 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptides and cross-linking agent to obtain cell adhesion gel.

[0070] (6) The cell adhesion gel was immersed in a solution containing 50 ng / mL vascular endothelial growth factor and 100 μg / mL anti-inflammatory drug. The growth factor and anti-inflammatory drug were allowed to enter the pores of the gel through diffusion adsorption. The gel was washed with PBS at pH 7.4 to remove unbound growth factor and then vacuum dried at 40°C for 4 h.

[0071] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 15 mmol / L and N-hydroxysuccinimide to a concentration of 8 mmol / L, and stir at 9°C for 14 h to crosslink the mixture to obtain medical recombinant collagen repair gel.

[0072] In step (1), the collagen solution concentration is 2 wt%; the chitosan solution concentration is 2 wt%; and the polylactic acid solution concentration is 10 wt%.

[0073] The crosslinking agent in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the nanocellulose, deionized water, matrix solution and crosslinking agent is 5:85:20:1.

[0074] The crosslinking agent in step (3) is N-hydroxysuccinimide; the foaming gas is carbon dioxide; the injection pressure of the foaming gas is 200 kPa and the injection time is 15 min.

[0075] The vacuum freeze-drying conditions in step (4) are -50℃, vacuum degree is 15Pa, and vacuum freeze-drying time is 48h.

[0076] The cross-linking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, cross-linking agent, base gel and PBS is 1:0.2:15:25.

[0077] In step (6), the mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS is 1:5:12.

[0078] Comparative Example 1

[0079] Based on Example 1, chitosan was not added to the raw materials, and the amount of polylactic acid added was changed to 31 parts, while the rest remained the same as in Example 1.

[0080] Comparative Example 2

[0081] Based on Example 1, polylactic acid was not added to the raw materials, and the amount of chitosan added was changed to 31 parts, while the rest remained the same as in Example 1.

[0082] Comparative Example 3

[0083] Based on Example 1, no foaming gas is injected, but everything else remains the same as in Example 1.

[0084] Comparative Example 4

[0085] Based on Example 1, step (4) is changed to freezing the gas-foamed gel solution at -80°C for 6.5 hours and then freeze-drying it under vacuum to obtain the base gel. The rest is the same as in Example 1.

[0086] Comparative Example 5

[0087] Based on Example 1, step (4) is changed to freezing the gas-foamed gel solution at -20°C for 6.5 hours and then freeze-drying it under vacuum to obtain the base gel. The rest is the same as in Example 1.

[0088] Performance testing:

[0089] Repair test: The medical recombinant collagen repair gels prepared in Application Examples 1-3 and Control Application Examples 1-5 were diluted to a 5% concentration solution using a culture medium containing 2% serum. The blank control group used a culture medium containing 2% serum, and the positive control group used a culture medium containing 10% serum.

[0090] Logarithmic growth phase BJ cells were seeded into 24-well plates at a density of 2.5 × 10⁻⁶. 5Cells / well. Incubate at 37°C in a CO2 incubator for 24 hours. Once the cells have reached near confluence, make a cross-shaped incision in each well using a pipette tip, then wash once with PBS. Add 500 μL of the diluted sample to each well and incubate at 37°C in a CO2 incubator until the predetermined observation time. At the set time, observe and measure the width of the scratches using a microscope.

[0091] The formula for calculating the scratch healing rate is as follows:

[0092]

[0093] Test results:

[0094]

[0095] The test results show that this invention improves tissue repair through the synergistic effect of collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drugs, and cell adhesion peptides. Furthermore, the gas foaming method, staged freezing, and freeze-drying facilitate cell penetration and tissue regeneration, further enhancing the repair effect.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A medical recombinant collagen repair gel, characterized in that: It includes the following ingredients by weight: 40-50 parts collagen, 15-20 parts chitosan, 10-15 parts polylactic acid, 5-10 parts nanocellulose, 1-2 parts vascular endothelial growth factor, 1-3 parts anti-inflammatory drugs, and 1-2 parts cell adhesion peptides. The preparation method of the aforementioned medical recombinant collagen repair gel includes the following steps: (1) Dissolve collagen in 0.1M glacial acetic acid solution and stir until completely dissolved to obtain collagen solution. Then dissolve chitosan in 0.1M glacial acetic acid solution and stir until transparent to obtain chitosan solution. Add collagen solution dropwise to chitosan solution and stir evenly to obtain mixed solution. Disperse polylactic acid in anhydrous ethanol and sonicate for 10-30 min to obtain polylactic acid solution. Add polylactic acid solution dropwise to mixed solution and stir evenly to obtain matrix solution. (2) Disperse nanocellulose in deionized water, sonicate for 10-20 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 6.5-7.4 with 1M sodium hydroxide solution, add crosslinking agent and stir evenly to obtain the basic gel solution; (3) Place the base gel solution in a sealed container, inject foaming gas through a micro gas injection device, add crosslinking agent to a concentration of 10-20 mmol / L, stir at 4℃ for 12 hours, and vacuum-treat at -10 to -100 kPa for 30 min to remove residual gas. (4) Freeze the gas-foamed gel solution at -80°C for 1-2 hours, then freeze it at -20°C for 4-6 hours, and then freeze-dry it under vacuum to obtain the base gel. (5) Add cross-linking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 20-40 min, add basic gel, soak for 3-5 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptides and cross-linking agent to obtain cell adhesion gel. (6) The cell adhesion gel was soaked in a solution containing 10-50 ng / mL vascular endothelial growth factor and 10-100 μg / mL anti-inflammatory drug. The growth factor and anti-inflammatory drug were allowed to enter the pores of the gel through diffusion adsorption. The gel was washed with PBS at pH 7.4 to remove unbound growth factor and then vacuum dried at 10-40℃ for 2-4 h. (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 5-15 mmol / L and N-hydroxysuccinimide to a concentration of 2-8 mmol / L, and stir at 1-9℃ for 10-14 h to crosslink the product to obtain medical recombinant collagen repair gel.

2. The recombinant collagen repair gel according to claim 1, characterized in that: The anti-inflammatory drug is any one of dexamethasone, indomethacin, and ibuprofen; the cell adhesion peptide is any one of RGD peptide and fibronectin.

3. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: The collagen solution concentration in step (1) is 1-2 wt%; the chitosan solution concentration is 1-2 wt%; and the polylactic acid solution concentration is 5-10 wt%.

4. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: The crosslinking agent in step (2) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution and crosslinking agent is 1-5:70-85:10-20:0.5-1.

5. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: The crosslinking agent in step (3) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the foaming gas is carbon dioxide or nitrogen; the injection pressure of the foaming gas is 50-200 kPa and the injection time is 5-15 min.

6. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: The vacuum freeze-drying conditions in step (4) are -50℃, vacuum degree is 5-15Pa, and vacuum freeze-drying time is 24-48h.

7. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: The crosslinking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or N-hydroxysuccinimide; the mass ratio of the cell adhesion peptide solution, crosslinking agent, base gel and PBS is 1:0.01-0.2:5-15:15-25.

8. The method for preparing a medical recombinant collagen repair gel according to claim 1, characterized in that: In step (6), the mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS is 1:3-5:8-12.

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