A collagen chitosan composite scaffold and its preparation method and application

Abstract

The invention discloses a collagen chitosan composite scaffold and its preparation method and application. The present invention obtains glycidyl methacrylate collagen by reacting collagen with glycidyl methacrylate; chitosan solution and methacrylic anhydride are reacted to obtain methacrylic chitosan; methacrylic anhydride and dopamine are reacted to obtain methacrylic dopamine; glycidyl methacrylate collagen, methacrylic chitosan, methacrylic dopamine and copper ion are mixed, and light curing reaction is carried out under the initiation of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate to obtain collagen chitosan composite scaffold. The scaffold has certain mechanical strength, toughness, fatigue resistance and viscoelasticity, and also has good biocompatibility, adhesion, antibacterial and antioxidant properties, and has outstanding application prospects in terms of acute wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of medical biomaterials, and in particular relates to a collagen-chitosan composite scaffold, a preparation method thereof, and an application thereof. Background Art

[0002] Tissue engineering aims to support and promote the regeneration of damaged or diseased tissues using scaffolds loaded with cells and growth factors. Tissue engineering scaffolds are expected to replace the extracellular matrix (ECM) and provide a temporary niche for cell growth. Therefore, the scaffold material should be strictly selected based on its biocompatibility, adhesion, mechanical properties, and other factors. Gelatin and chitosan (CS) are the preferred natural biomaterials for tissue engineering scaffolds because they are closely related to the ECM. Gelatin is a hydrolysis product of collagen, the main component of the ECM. It has been widely used in tissue engineering due to its antigenicity and biocompatibility. However, its low mechanical strength and adhesion properties limit its application. Chitosan is a polysaccharide made by deacetylation of chitin with good antibacterial properties. Due to its excellent biological properties, mixtures of gelatin and CS have been used to manufacture various scaffolds, such as skin, bone, nerve, and liver.

[0003] Although gelatin and CS-based hydrogels have their advantages, their poor mechanical properties have become a major obstacle limiting their application as scaffolds. In order to improve the mechanical strength of this scaffold, chemical crosslinking agents such as carbodiimide and glutaraldehyde are often added. However, this method will cause unreacted crosslinking agents to remain inside the scaffold, resulting in toxic reactions and is not suitable for use in wounds. Therefore, the method of using ultraviolet light crosslinking to enhance the mechanical strength of the scaffold without using a crosslinking agent has attracted widespread attention. For example, Ji et al. developed a dual-crosslinked hydrogel based on gelatin methacryloyl and sulfhydrylated chitosan [Ji, S., et al., A Dual-Crosslinked Hydrogel Based on Gelatin Methacryloyl and Sulfhydrylated Chitosan for Promoting Wound Healing. International Journal of Molecular Sciences, 2023. 24 (3): p. 2447]. The compression modulus of this hydrogel can reach 31.78 ± 3.315 kPa, with high rigidity and compressive resistance; Ana Isabel et al. prepared a biocompatible scaffold composed of chemically crosslinked chitosan and gelatin [Canas, AI, JP Delgado, and C. Gartner, Biocompatible scaffolds composed of chemically crosslinked chitosan and gelatin for tissue engineering. Journal of Applied Polymer Science, 2016. 133 (33).], and the compression modulus of this crosslinked scaffold is in the range of 0.416–2.216 MPa; National Invention Patent Application CN108047465A discloses a methacrylate gelatin / chitosan interpenetrating network hydrogel, which has the characteristics of good biocompatibility, non-toxicity, and excellent mechanical properties, and can be used as a material for tissue repair. Although the above-mentioned prior art has optimized the preparation of gelatin / chitosan composite scaffolds and significantly improved their mechanical properties, if this scaffold is used in the field of skin healing, then although the high mechanical properties have resistance to external compression, it will inevitably weaken its fatigue resistance and toughness. Therefore, in order for the scaffold to perform better in wound healing, the scaffold material is required to have properties such as rigidity, toughness and viscoelasticity to provide sufficient protection for wound healing.

[0004] Therefore, the gel still needs to be further optimized to overcome the above-mentioned shortcomings. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a method for preparing a collagen-chitosan composite scaffold.

[0006] Another object of the present invention is to provide a collagen-chitosan composite scaffold obtained by the above preparation method.

[0007] Another object of the present invention is to provide an application of the above-mentioned collagen-chitosan composite scaffold.

[0008] The purpose of the present invention is achieved through the following technical solution: A method for preparing a collagen chitosan composite scaffold comprises the following steps:

[0009] (1) Preparation of glycidyl methacrylate collagen (rCol-GMA): Dissolve collagen in pure water to form a collagen solution; Mix the collagen solution with glycidyl methacrylate (GMA) at a volume ratio of 1:0.5 to 1, then dialyze in pure water, and freeze-dry after dialysis to obtain glycidyl methacrylate collagen;

[0010] (2) Preparation of methacrylate chitosan (CSMA): dissolving chitosan in an acetic acid solution to obtain a chitosan solution; mixing the chitosan solution with methacrylic anhydride, reacting, dialyzing, and freeze-drying to obtain methacrylate chitosan;

[0011] (3) Preparation of dopamine methacrylate (DMA):

[0012] A. Dissolve sodium tetraborate and sodium bicarbonate in deionized water, add dopamine hydrochloride after bubbling with nitrogen, and mix well under nitrogen stirring to obtain a dopamine solution;

[0013] B. deoxygenating tetrahydrofuran and uniformly mixing it with methacrylic anhydride to obtain a methacrylic anhydride solution;

[0014] C. Add the methacrylic anhydride solution dropwise to the dopamine solution, maintain the pH value of the resulting system at 8-10, and stir to react;

[0015] D. Extract the obtained reaction product with ethyl acetate, retain the aqueous layer, and adjust the pH value to 1-4;

[0016] E. Continue to extract with ethyl acetate, retain the organic layer, remove the solvent, and obtain dopamine methacrylate;

[0017] (4) Preparing a collagen chitosan composite scaffold: dissolving the methacrylated collagen obtained in step (1) in pure water to obtain a methacrylated collagen solution; dissolving the methacrylated chitosan obtained in step (2) in acetic acid solution to obtain a methacrylated chitosan solution; dissolving the methacrylated dopamine obtained in step (3) in dimethyl sulfoxide to obtain a methacrylated dopamine solution; mixing the methacrylated collagen solution and the methacrylated chitosan solution to obtain a mixed solution A; mixing copper ions with the methacrylated dopamine solution to obtain a mixed solution B; mixing the mixed solutions A and B, adding phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) solution and stirring evenly to obtain a reaction system, and then performing a photocuring reaction to obtain a collagen chitosan composite scaffold.

[0018] The collagen described in step (1) is preferably recombinant human collagen; more preferably recombinant human type I collagen (rCol); most preferably recombinant human type I collagen having an amino acid sequence as shown in SEQ ID NO.1.

[0019] The recombinant human type I collagen is obtained by combining a fibroblast adhesion sequence (GERGDLGPQGIAGQRGVVGERGERGERGAS) and an integrin α2β1 recognition sequence (GFPGER), has a size of 120 kDa, and has good biocompatibility and adhesion.

[0020] The concentration of the collagen solution in step (1) is preferably 80 to 120 μg / mL; more preferably 100 μg / mL.

[0021] The collagen solution in step (1) and the glycidyl methacrylate are preferably mixed in a volume ratio of 1:0.6.

[0022] The reaction conditions in step (1) are preferably stirred at 2-8°C for 8±2 hours.

[0023] The dialysis time in step (1) is preferably 5±2 days.

[0024] The concentration of the acetic acid solution in step (2) is preferably 2-4% by volume; more preferably 3% by volume.

[0025] The dissolution time in step (2) is preferably 24±12 hours.

[0026] The concentration of the chitosan solution in step (2) is preferably 2-4% (w / v); more preferably 3% (w / v).

[0027] The amount of methacrylic anhydride used in step (2) is excessive relative to chitosan, preferably calculated based on a volume ratio of methacrylic anhydride:chitosan solution = 3:1.

[0028] The reaction conditions in step (2) are preferably room temperature for 3±1 hours.

[0029] The room temperature is 10-40°C, preferably 20-30°C, and more preferably 24-26°C.

[0030] The dialysis time in step (2) is preferably 5±2 days.

[0031] The sodium tetraborate and the sodium bicarbonate described in step (3)A are preferably mixed in a mass ratio of 5:2.

[0032] The amount of dopamine hydrochloride used in step (3) A is sodium tetraborate: dopamine hydrochloride = mass ratio of 2:1.

[0033] The bubbling time in step (3)A is preferably 60±20 minutes.

[0034] The tetrahydrofuran described in step (3) B is a reaction medium and does not participate in the reaction. Its amount is preferably 4 to 6 times the volume of methacrylic anhydride.

[0035] The amount of methacrylic anhydride used in step (3) B is excessive relative to dopamine hydrochloride, preferably in a mass ratio of dopamine hydrochloride: methacrylic anhydride = 10:9.5-10.

[0036] The pH value in step (3)C is preferably adjusted by using a deoxygenated 1.0 M NaOH solution.

[0037] The reaction conditions described in step (3)C are preferably stirring at room temperature for 15±5 hours.

[0038] The room temperature is 10-40°C, preferably 20-30°C, and more preferably 24-26°C.

[0039] The number of extractions in step (3)D is preferably 1.

[0040] The pH value described in step (3)D is preferably 2-3.

[0041] The pH value in step (3)D is preferably adjusted by 6M HCl solution.

[0042] The number of extractions in step (3)E is preferably 2 times.

[0043] The specific step of removing the solvent in step (3)E is preferably: drying with anhydrous magnesium sulfate and then distilling under reduced pressure.

[0044] The concentration of the glycidyl methacrylate collagen solution in step (4) is preferably 4 wt %.

[0045] The concentration of the acetic acid solution in step (4) is preferably 0.5% (v / v).

[0046] The concentration of the methacrylic acid chitosan solution in step (4) is preferably 4 wt %.

[0047] The concentration of the dopamine methacrylate solution in step (4) is preferably 8.225%.

[0048] The copper ions described in step (4) are derived from soluble copper salts, preferably copper chloride.

[0049] The copper ions in step (4) and the dopamine methacrylate are preferably mixed in a molar ratio of 1:1 to 3; more preferably in a molar ratio of 1:2.

[0050] In the reaction system described in step (4), glycidyl methacrylate collagen, chitosan methacrylate and dopamine methacrylate are mixed in a mass ratio of 16:16:16.45 to 65.8.

[0051] The concentration of LAP in the reaction system in step (4) is preferably 0.01 to 0.1 wt %; more preferably 0.05 wt %.

[0052] The light curing conditions in step (4) are preferably irradiation under ultraviolet light for 10 to 30 seconds; more preferably irradiation under ultraviolet light for 15 seconds.

[0053] The wavelength of the ultraviolet light is preferably 365 nm.

[0054] The power of the ultraviolet light is preferably 1w / cm 2 .

[0055] A collagen-chitosan composite scaffold, obtained by the above-mentioned preparation method, is a hydrogel scaffold with rigidity, toughness, and viscoelasticity, and can solve the problem of the current gelatin / chitosan composite scaffold having too high mechanical properties and poor fatigue resistance.

[0056] The application of the above collagen chitosan composite scaffold in the preparation of tissue repair materials or tissue engineering scaffolds in the field of tissue engineering.

[0057] The present invention has the following advantages and effects compared to the prior art:

[0058] The present invention first replaces gelatin with collagen, which has better tissue compatibility and adhesion properties than gelatin. At the same time, chitosan and recombinant collagen are methacrylated to achieve covalent cross-linking under ultraviolet light. At the same time, a methacrylated dopamine-copper system is introduced, so that the double-network cross-linking system generated by the entire scaffold can reduce its rigidity, so that it not only has a certain mechanical strength, but also can resist a certain external pressure, while increasing its toughness, fatigue resistance and viscoelasticity.

[0059] Furthermore, the recombinant collagen used in the present invention not only avoids the immunogenicity and cross-infection risks of animal-derived collagen, but also has better biocompatibility with wound surfaces compared to gelatin.

[0060] Furthermore, the present invention uses recombinant human type I collagen containing a large amount of integrin and fibroblast adhesion sequences, which has good cell adhesion performance and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1This is a small-scale expression graph of recombinant human type I collagen; lane M is a protein marker (ThermoFisher #26616). Lane W is the whole fluid after bacterial cell disruption, lane S is the supernatant, and lane P is the precipitate.

[0062] Figure 2 The figure shows the results of large-scale expression and purification of recombinant human type I collagen; lane M is the protein marker (Thermo Fisher #26616), and lanes I-4 are the purified recombinant human type I collagen.

[0063] Figure 3 This is a comparison chart of the adhesion between recombinant collagen and gelatin.

[0064] Figure 4 It is a composite of methacrylate chitosan, glycidyl methacrylate recombinant human type I collagen and methacrylate dopamine 1 H-NMR spectrum.

[0065] Figure 5 This is a diagram of the inverted gel of the recombinant human type I collagen / chitosan composite scaffold.

[0066] Figure 6 This is the stress-strain curve of the recombinant human type I collagen / chitosan composite scaffold.

[0067] Figure 7 This is the compression modulus diagram of the recombinant human type I collagen / chitosan composite scaffold.

[0068] Figure 8 This is the compression-cycling curve of the recombinant human type I collagen / chitosan composite scaffold.

[0069] Figure 9 This is a viscosity test diagram of the recombinant human type I collagen / chitosan composite scaffold.

[0070] Figure 10 Diagram of wound healing using recombinant human type I collagen / chitosan composite scaffold as tissue repair material.

[0071] Figure 11 This is a graph showing the wound healing rate of recombinant human type I collagen / chitosan composite scaffolds used as tissue repair materials in wounds. DETAILED DESCRIPTION

[0072] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0073] Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment. Unless otherwise specified, the materials and reagents used in the present invention are all commercially available.

[0074] Example 1: Preparation of recombinant human type I collagen

[0075] The amino acid sequence of recombinant human type I collagen is SEQ ID No. 1. The gene sequence was optimized according to the codon preference of the host Escherichia coli expression. The optimized nucleotide sequence is shown in SEQ ID No. 2. The sequence is constructed in a HisTrap containing two restriction sites, NdeⅠ and KpnⅠ. TM The recombinant human type I collagen was purified by HP Column onto the pET28a vector and transformed into Escherichia coli BL21 (DE3) for expression. The expression and purification of recombinant human type I collagen was carried out according to conventional protein expression methods:

[0076] (1) Seed liquid preparation: Pick a single colony from the plate in a clean bench and inoculate it into 10 mL of LB liquid medium (Kanam resistance, Kan + ) and cultured in a constant temperature shaker at 37°C and 220 rpm for 10 to 12 h as seed solution.

[0077] (2) Protein expression and purification: The above seed solution was transferred to two shake flasks (Kan + ), where shake flask No. 1 is marked as uninduced and shake flask No. 2 is marked as induced. After culturing at 37°C and 220 rpm in a constant temperature shaker for 2-3 hours, the OD value of the bacterial solution was measured. 600 When the value is 0.8, add IPTG (isopropyl-β-D-thiogalactopyranoside) with a final concentration of 1mM to the shake flask No. 2 to induce collagen expression and continue to culture for 5h. After the induction is completed, measure the OD of the bacterial solution 600 The supernatant was verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after the cells were broken. The results were as follows: Figure 1 As shown, when IPTG was added (shake flask No. 2), collagen was induced to express. The bacterial cells in shake flask No. 2 were collected and purified according to the following method to obtain recombinant human collagen.

[0078] 1) Resuspend the cells in Buffer A at 10 OD / mL and disrupt the cells using a high-pressure disruptor (temperature: 4°C, pressure: 22 kpsi). Centrifuge at 4°C, 10,000 rpm for 30 min to obtain the supernatant and precipitate. The recombinant protein is present in the supernatant, which is collected and filtered through a 0.22 μm filter to obtain a pre-column sample.

[0079] 2) GE's (HisTrap TM HP Column, 5mL) pre-packed column was used as the purification medium for one-step affinity purification. The AKTA chromatography system was flushed with ultrapure water at a flow rate of 5mL / min until the UV baseline was stable. The column was loaded and the nickel column was equilibrated with Buffer A. The pre-column sample was injected at a rate of 3mL / min. During this period, the post-column effluent sample (this is the flow-through) was collected. After the loading was completed, it was rinsed with Buffer A and then linearly eluted with Buffer B at 5mL / min. When UV 280 The sample was collected when the peak appeared and stopped after the peak appeared. The purified sample was tested by SDS-PAGE electrophoresis to detect the protein molecular weight and purity (such as Figure 2 The purity of the prepared recombinant human collagen was 91.82%. The obtained recombinant human collagen was dialyzed in a 1×PBS solution using a dialysis bag with a pore size of 14 kD. After dialysis, the recombinant human collagen was freeze-dried in a freeze dryer to obtain recombinant collagen (rCol).

[0080] Buffer A: 20 mmol / L Tris-HCl, 20 mmol / L imidazole, 500 mmol / L NaCl, pH 8.5. Remove impurities by filtering with a 0.22 μm filter membrane, degas by ultrasonication (before use), and store at room temperature.

[0081] Buffer B preparation: 20 mmol / L Tris-HCl, 500 mmol / L imidazole, 500 mmol / L NaCl, pH 8.5. Filter through a 0.22 μm filter membrane to remove impurities, degas by ultrasonication (before use), and store at room temperature.

[0082] Example 2: Adhesion test of recombinant human type I collagen

[0083] High (100 μg / mL), medium (50 μg / mL), and low (12.5 μg / mL) concentrations of recombinant human type I collagen and gelatin were evenly plated on a 96-well plate without TC treatment. After drying in a cell culture incubator, recombinant human type I collagen membranes and gelatin membranes were formed. Mouse embryonic fibroblast (NIH / 3T3) cell suspensions were plated at 5×10 4 Cells were seeded into 96-well plates containing recombinant type I collagen membrane and gelatin membrane. After culturing for 4 hours, cell adhesion was detected using a CCK-8 assay. Wells without collagen membrane were used as controls. Three composite wells were set for each concentration. Figure 3 As shown in the figure, recombinant human type I collagen has better adhesion properties than gelatin.

[0084] Example 3: Preparation of recombinant human type I collagen / chitosan composite scaffold

[0085] (1) Recombinant human type I collagen was first dissolved in pure water to obtain a recombinant human type I collagen solution with a concentration of 100 μg / mL; the recombinant human type I collagen solution was mixed with glycidyl methacrylate (GMA), wherein the recombinant human type I collagen solution and glycidyl methacrylate were mixed in a volume ratio of 1:0.6, reacted in a 4°C refrigerator for 8 h, dialyzed in pure water for 4 days, and freeze-dried to obtain glycidyl methacrylate collagen (rCol-GMA).

[0086] (2) Chitosan was dissolved in 3% (v / v) acetic acid at room temperature for 24 hours to obtain a 3% (w / v) chitosan solution; methacrylic anhydride was then added, wherein the chitosan solution and methacrylic anhydride were mixed in a volume ratio of 1:3, and the mixture was reacted for 4 hours, dialyzed in pure water for 6 days, and freeze-dried to obtain methacrylate chitosan (CSMA).

[0087] (3) Dissolve 20 g of sodium tetraborate and 8 g of sodium bicarbonate in deionized water, bubble with nitrogen for 60 min, add 10 g of dopamine hydrochloride to the mixture, and stir under nitrogen to obtain a dopamine solution. Take 50 mL of tetrahydrofuran solution, deoxygenate it, and add 9.4 mL of methacrylic anhydride (density 1.035 g / cm 3 ) to obtain a mixed solution. The mixed solution was gradually added dropwise to the dopamine solution, and the pH was adjusted by adding deoxygenated 1.0M NaOH. The pH was periodically maintained at 8.5-9. After stirring at room temperature for 17 hours, the solution was extracted once with ethyl acetate, retaining the aqueous layer. The pH was then adjusted to 2 with 6M HCl, and the mixture was extracted twice with ethyl acetate, retaining the organic layer. After drying over anhydrous magnesium sulfate, the product was distilled under reduced pressure to obtain dopamine methacrylate (DMA).

[0088] (4) The obtained glycidyl methacrylate collagen, methacrylate chitosan, and methacrylate dopamine were dissolved in D2O (heavy water), D2O containing 1% trifluoroacetic acid, and DMSO-d6 (deuterated dimethyl sulfoxide), respectively. 1 H-NMR confirmed whether the methacrylic acid group was successfully introduced into the side chain. Figure 4 .

[0089] (5) CSMA dry powder was dissolved in 0.5% (v / v) acetic acid to prepare a 4wt% solution, rCol-GMA dry powder was dissolved in pure water to prepare a 4wt% solution, DMA powder was dissolved in 0.5% (v / v) dimethyl sulfoxide (DMSO) to prepare an 8.225wt% solution, CuCl2 powder was dissolved in pure water to prepare a 10wt% solution, and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) powder was dissolved in pure water to prepare a 1wt% solution. Then, according to the formula in Table 1, the above solutions were mixed to obtain CS / rCol, CS / rCol / Cu, and the like. 50 / DMA、CS / rCol / Cu 100 / DMA and CS / rCol / Cu 200 / DMA four groups of hydrogel scaffold precursor solutions were exposed to ultraviolet light (365nm, 1w / cm 2 ) was irradiated for 15 seconds to form a methacrylated recombinant human type I collagen / chitosan composite hydrogel scaffold, such as Figure 5 shown.

[0090] Table 1: Preparation parameters of recombinant human type I / chitosan scaffolds

[0091]

[0092] Example 4: Compression test of recombinant human type I collagen / chitosan composite scaffold

[0093] The compressive stress-strain measurements of methacrylated recombinant human type I collagen / chitosan composite scaffolds were performed using a universal testing machine (DMA Q800, TA, USA) with the pressure ranging from 1.0000 N / min to 3.0000 N. Figure 6 As shown in the stress-strain curve, when reaching the same strain, the CS / rCol scaffold uses a larger stress and has a higher rigidity, while the scaffold containing the dopamine methacrylate-copper system uses a smaller stress and has a weaker rigidity. In addition, it was clearly found during the test that when the strain reached above 60%, the CS / rCol scaffold had already broken, while the scaffold containing the dopamine methacrylate-copper system had not yet broken, and even remained in its original shape after the stress was removed. The strain range of 5%-25% in the above stress-strain curve was selected for plotting, and the following was obtained: Figure 7 The compression modulus diagram quantitatively shows that after replacing gelatin with recombinant human type I collagen, the compression modulus of the CS / rCol scaffold is higher, while the compression modulus of the scaffold introduced with the dopamine methacrylate-copper system is significantly reduced.

[0094] Example 5: Compression-Cycling Test of Recombinant Human Type I Collagen / Chitosan Composite Scaffold

[0095] The compressive stress-strain measurement of methacrylated recombinant human type I collagen / chitosan composite scaffold was continued to be tested using a universal testing machine (DMAQ800, TA, USA) through 20 cycles of loading-unloading pressure. Figure 8 As shown, the CS / rCol scaffold exhibits greater energy dissipation, while the scaffold incorporating the dopamine methacrylate-copper system exhibits lower energy dissipation and exhibits improved viscoelasticity and fatigue resistance. This is likely due to the incorporation of the dopamine methacrylate-copper system, which creates a double network crosslink. These networks intertwine and entangle with each other, allowing for a more dispersed transfer of stress to the other network when subjected to external pressure.

[0096] Example 6: Viscosity test of recombinant human type I collagen / chitosan composite scaffold

[0097] The rheological behavior of the hydrogel was tested using a TA rheometer (HR30, WATERS, USA). The measured rheological parameters were the storage modulus (elastic modulus, G') and the loss modulus (viscous modulus, G'). The test was performed in an oscillation frequency sweep test in the shear rate range of 0.1 rad / s to 500. The results are shown in Figure 2. Figure 9 It was shown that the CS / rCol scaffold itself was sticky, and its stickiness was improved to a certain extent after the introduction of the dopamine methacrylate-copper system.

[0098] Example 7: Application of recombinant human type I collagen / chitosan composite scaffold as tissue repair material in wound healing

[0099] Female SD rats (180-220 g, 4-6 weeks old) purchased from Guangzhou Sijia Jingda Biotechnology Co., Ltd. were selected to establish a full-thickness skin defect model and conduct an in vivo wound healing experiment (AEC No.: 2019053). They were divided into a control group (no treatment), a positive control group (purchased Extra Thin CGF TM Dressing hydrocolloid dressing), CS / rCol / Cu 100 / DMA experimental group. Anesthesia was performed using isoflurane inhalation anesthesia, and the backs of the rats were shaved one day before the start of the experiment. A full-thickness circular skin wound with a diameter of 2 cm was constructed on the back of each rat. The wounds were treated according to the groups, and all wounds were photographed on days 0, 3, 7, 14, and 21. The wound boundaries were drawn using ImageJ software, and the wound area was calculated. Generally speaking, wound size is the most intuitive indicator for evaluating wound healing rate, and is used to compare the differences in therapeutic effects between the composite hydrogel and the control group. Figure 10 and 11As shown, in the first 7 days of wound healing, the hydrogel in the experimental group showed better healing effect than the control group and the positive control group. The wound had healed to 28.6% of its initial size on the 7th day, and the cure rate was higher than that of the control group (59.3%) and the positive control group (44.2%). However, since the present invention only uses the composite hydrogel at the initial stage of the wound, rather than repeatedly using it, the healing rate in the later stage of wound healing is not significantly different from that of the positive control group and the control group. However, the characteristic of the composite hydrogel in promoting rapid wound healing in a short time makes it of great significance in the clinical treatment of acute wounds.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a collagen chitosan composite scaffold, characterized in that The steps include: (1) Preparation of glycidyl methacrylate collagen: dissolving collagen in pure water to form a collagen solution; mixing the collagen solution with glycidyl methacrylate in a volume ratio of 1:0.5-1, followed by dialysis in pure water, and freeze-drying to obtain glycidyl methacrylate collagen; (2) Preparation of methacrylate chitosan: dissolving chitosan in acetic acid solution to obtain chitosan solution; mixing the chitosan solution with methacrylic anhydride, reacting, dialyzing, and freeze-drying to obtain methacrylate chitosan; (3) Preparation of dopamine methacrylate: A. Dissolve sodium tetraborate and sodium bicarbonate in deionized water, add dopamine hydrochloride after bubbling with nitrogen, and mix well under nitrogen stirring to obtain a dopamine solution; B. deoxygenating tetrahydrofuran and uniformly mixing it with methacrylic anhydride to obtain a methacrylic anhydride solution; C. Add the methacrylic anhydride solution dropwise to the dopamine solution, maintain the pH value of the resulting system at 8-10, and stir to react; D. Extract the obtained reaction product with ethyl acetate, retain the aqueous layer, and adjust the pH value to 1-4; E. Continue to extract with ethyl acetate, retain the organic layer, remove the solvent, and obtain dopamine methacrylate; (4) Preparing a collagen chitosan composite scaffold: dissolving the methacrylate glycidyl collagen obtained in step (1) in pure water to obtain a methacrylate glycidyl collagen solution; dissolving the methacrylate chitosan obtained in step (2) in acetic acid solution to obtain a methacrylate chitosan solution; dissolving the methacrylate dopamine obtained in step (3) in dimethyl sulfoxide to obtain a methacrylate dopamine solution; mixing the methacrylate glycidyl collagen solution and the methacrylate chitosan solution to obtain a mixed solution A; mixing copper ions with the methacrylate dopamine solution to obtain a mixed solution B; mixing the mixed solutions A and B, adding phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution and stirring evenly to obtain a reaction system, and then performing a photocuring reaction to obtain a collagen chitosan composite scaffold.

2. The method for preparing the collagen-chitosan composite scaffold according to claim 1, wherein: The collagen described in step (1) is recombinant human collagen.

3. The method for preparing the collagen-chitosan composite scaffold according to claim 2, characterized in that: The collagen described in step (1) is recombinant human type I collagen.

4. The method for preparing the collagen-chitosan composite scaffold according to claim 3, wherein: The collagen described in step (1) is a recombinant human type I collagen having an amino acid sequence as shown in SEQ ID NO.

1.

5. The method for preparing the collagen-chitosan composite scaffold according to claim 1, wherein: The reaction conditions in step (1) are stirring at 2-8°C for 8±2 hours; The duration of dialysis described in step (1) is 5 ± 2 days.

6. The method for preparing the collagen-chitosan composite scaffold according to claim 1, characterized in that: The concentration of the acetic acid solution in step (2) is 2 to 4% by volume; The concentration of the chitosan solution in step (2) is 2-4%, w / v; The amount of methacrylic anhydride used in step (2) is calculated based on a volume ratio of methacrylic anhydride to chitosan solution of 3:

1. The reaction conditions in step (2) are as follows: reaction at room temperature for 3±1 hours; The dialysis time described in step (2) is 5±2 days.

7. The method for preparing the collagen-chitosan composite scaffold according to claim 1, characterized in that: The sodium tetraborate and the sodium bicarbonate described in step (3) A are mixed in a mass ratio of 5:2; The amount of dopamine hydrochloride in step (3) A is sodium tetraborate: dopamine hydrochloride = mass ratio 2:1; The amount of methacrylic anhydride used in step (3) B is in the ratio of dopamine hydrochloride to methacrylic anhydride = 10:9.5-10 by mass.

8. The method for preparing the collagen-chitosan composite scaffold according to claim 1, characterized in that: The bubbling time in step (3) A is 60±20 minutes; The volume of tetrahydrofuran in step (3) B is 4 to 6 times that of methacrylic anhydride; The pH value described in step (3) C is adjusted by using a deoxygenated 1.0 M NaOH solution; The reaction conditions described in step (3) C are stirring at room temperature for 15±5 hours; The number of extractions in step (3) D is 1; The pH value in step (3) D is 2 to 3; The pH value in step (3) D is adjusted by 6 M HCl solution; The number of extractions in step (3) E is 2 times; The specific steps of removing the solvent in step (3) E are: drying with anhydrous magnesium sulfate and then distilling under reduced pressure.

9. The method for preparing the collagen-chitosan composite scaffold according to claim 1, characterized in that: The copper ions described in step (4) are derived from soluble copper salts; The copper ions described in step (4) and the dopamine methacrylate are mixed in a molar ratio of 1:1 to 3; In the reaction system described in step (4), glycidyl methacrylate collagen, chitosan methacrylate and dopamine methacrylate are mixed in a mass ratio of 16:16:16.45-65.8; The concentration of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt in the reaction system in step (4) is 0.01 to 0.1 wt%; The light curing condition described in step (4) is irradiation under ultraviolet light for 10 to 30 seconds.

10. The method for preparing the collagen-chitosan composite scaffold according to claim 9, characterized in that: The wavelength of the ultraviolet light is 365nm; The power of the ultraviolet light is 1 w / cm 2 .

11. A collagen-chitosan composite scaffold, characterized by: The method is obtained by the preparation method according to any one of claims 1 to 10.

12. Use of the collagen-chitosan composite scaffold according to claim 11 in preparing tissue repair materials or tissue engineering scaffolds.

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