Efficient anti-infection double-layer artificial skin and preparation method thereof

Abstract

The invention discloses a high-efficiency anti-infection double-layer artificial skin and a preparation method thereof. The high-efficiency anti-infection double-layer artificial skin has an excellent antibacterial effect and can reduce the occurrence of mucous membrane and instrument related infections. The collagen scaffold is a recombinant collagen scaffold with a three-dimensional pore structure, provides a suitable physical structure for cells, is beneficial to adhesion and proliferation of fibroblasts, vascular endothelial cells and the like, and has a remarkable skin wound repair effect. According to the invention, mTGase is mainly used for cross-linking glutamine and lysine sites to form protein-protein cross-linking; the Bis-PEG6-Acid can be used for cross-linking the Lys (Lys-NH2) and another protein, so as to form PEG (Polyethylene Glycol)-protein cross-linking; the mTGase and the Bis-PEG6-Acid do not directly and synergistically catalyze the same reaction site, a primary protein network is firstly constructed by the mTGase, the crosslinking density is further enhanced by the Bis-PEG6-Acid, a double-network structure is realized, and the mechanical strength, the degradation resistance and the hydrophilicity of the stent material are enhanced. And the hydrogel also has good tensile elasticity and biocompatibility, and is free of cytotoxicity and sensitization.

Description

Technical Field

[0001] This invention relates to the field of artificial skin, and more specifically to a highly effective anti-infection double-layer artificial skin. Background Technology

[0002] Collagen is an important protein in the human body, widely found in tissues such as skin, tendons, and cartilage. Due to its low immunogenicity, biodegradability, and good biocompatibility, collagen is now widely used as a raw material for implantable medical devices in clinical fields such as wound repair and other tissue engineering.

[0003] Deep wounds often involve damage to the entire thickness of the skin, including the epidermis, dermis, and subcutaneous tissue. Restoring the structure and function of these layers requires precise tissue engineering techniques and biomaterials. Current clinical treatments often cannot fully mimic the complex structure and function of natural skin. For example, existing skin wound repair products, including decellularized matrix products, polysaccharide gels, and processed products derived from extracted collagen, are mainly animal-derived medical devices. Their raw materials are extracted from animal tissues, leading to quality variations, risks of animal viruses and immune rejection, and poor angiogenesis effects. Furthermore, deep wounds heal slowly, have weakened local immune protection, and are prone to infection with prolonged exposure, affecting wound prognosis and, in severe cases, even endangering the patient's life. Summary of the Invention

[0004] To overcome the aforementioned problems, the present invention aims to provide a highly efficient anti-infection bilayer artificial skin and its preparation method. The objective of this invention is to produce a highly efficient anti-infection bilayer artificial skin, a recombinant collagen scaffold with high cell proliferation and migration effects and a three-dimensional porous structure, providing cells with a suitable physical structure (100-300 μm), facilitating the adhesion and proliferation of fibroblasts, vascular endothelial cells, etc., and exhibiting significant skin wound repair effects. It also possesses significant antibacterial properties, reducing the occurrence of mucosal and instrument-related infections. The method of this invention has excellent modification effects (complementary cross-linking sites). mTGase first constructs a primary protein network, and Bis-PEG6-Acid further enhances the cross-linking density, achieving a bilayer network structure. This ultimately enhances the mechanical strength, degradation resistance, and water absorption of the scaffold material. The cross-linking agents are highly safe (enzymes and PEG derivatives), and the bilayer cross-linking form requires only small amounts, resulting in a material with good tensile elasticity. It exhibits good biocompatibility and is free of cytotoxicity and sensitization.

[0005] This invention discloses a two-layer artificial dermal repair material. The upper layer has a dual-effect antibacterial coating, which can reduce the risk of infection in chronic large-area wounds. The lower layer, a recombinant humanized collagen scaffold, is three-dimensionally porous, providing a suitable spatial structure for cell growth, promoting cell proliferation and migration, accelerating vascularization, and improving wound healing efficiency. The cross-linking method used is cross-crosslinking, which does not directly synergistically catalyze the same reaction site, resulting in a more three-dimensional cross-linking effect. Furthermore, while ensuring mechanical strength, the dosage of cross-linking agent is reduced (preferably, the EDC concentration is 0.01-1%, and the PEG linker concentration is 0.1-1%), improving the material's biocompatibility.

[0006] To achieve the above objectives, according to some technical solutions of the present invention, a method for preparing a highly effective anti-infection double-layer artificial skin is provided, characterized in that the preparation method comprises the following steps: Step 1: Prepare a chitosan silver ion solution, coat the chitosan silver ion solution onto an inert polymer film to obtain the upper layer structure of a double-layer artificial skin; Step 2: Prepare a recombinant collagen solution, add transglutaminase (mTGase), react, dry, and obtain mTGase-crosslinked recombinant collagen scaffold material. Step 3: Prepare Bis-PEG6-Acid solution, place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution for a second crosslinking, and wash with purified water after the reaction.

[0007] Step 4: The cleaned material is laid flat on the inert polymer material film prepared in Step 1, with the contact side being a chitosan coating loaded with silver ions. The entire material is then dried to obtain a highly effective anti-infection double-layer artificial skin.

[0008] According to some of the technical solutions of the present invention, a method for preparing a highly effective anti-infection double-layer artificial skin is provided. In step 1 above, the inert polymeric material membrane is a polyurethane membrane, silicone or polyvinyl alcohol, the silver ion source is silver sulfate, the chitosan concentration is 0.5%-2.5%, the silver content is 1-3% of the chitosan mass, and the chitosan silver ion solution is coated onto the inert polymeric material membrane at a rate of 0.2-0.5 ml per square centimeter; in this step, the silver mass is calculated as silver ions.

[0009] In this invention, the preferred inert polymeric membrane material is a medical polyurethane membrane.

[0010] According to some of the technical solutions of the present invention, a method for preparing a highly effective anti-infection double-layer artificial skin is provided, wherein in step 2 above, the concentration of the recombinant collagen solution is 1-15%, and 0.05 to 0.2 U / mL of transglutaminase (mTGase) is added, with a pH of 6.0 to 7.0.

[0011] According to some of the technical solutions of the present invention, a method for preparing a highly effective anti-infection double-layer artificial skin is provided, wherein the preparation of Bis-PEG6-Acid solution in step 3 above is the preparation of EDC.HCl / Bis-PEG6-Acid solution.

[0012] According to some of the technical solutions of the present invention, a method for preparing a highly effective anti-infection double-layer artificial skin is provided. In step 3 of the above preparation method, an EDC.HCl / Bis-PEG6-Acid solution is prepared, with an EDC concentration of 0.01-1% and a PEG linker concentration of 0.1-1%. The reaction temperature is 25-45℃, and the reaction time is 4-20h. After the reaction, the material is wrapped in a dialysis bag and washed with purified water in a shaker at a speed of 60-100rpm. The drying method in the above preparation method is freeze drying.

[0013] According to some of the technical solutions of the present invention, a highly effective anti-infection double-layer artificial skin is provided, which is prepared according to any of the above methods.

[0014] According to some of the technical solutions of the present invention, a highly effective anti-infection double-layer artificial skin is provided. The double-layer artificial skin has a double-layer structure comprising an upper layer and a lower layer. The upper layer is a polyurethane membrane, silicone, or polyvinyl alcohol. The inner side of the upper layer is a chitosan coating loaded with silver ions. The lower layer is a cross-linked recombinant humanized collagen dermal matrix material. The cross-linked recombinant humanized collagen dermal matrix material is a recombinant collagen scaffold material cross-linked with mTGase and cross-linked with Bis-PEG-Acid. The double-layer artificial skin has a three-dimensional porous structure.

[0015] This invention solves the problem of low antibacterial properties in some existing technologies and does not use sulfuric acid. This invention achieves the bonding of the double-layer structure through the self-adhesive properties of chitosan, and the overall antibacterial effect and the repair effect of the lower collagen layer are significantly improved compared with the existing technologies.

[0016] According to some of the technical solutions of the present invention, a highly effective anti-infection double-layer artificial skin is provided, wherein the silver ions in the above-mentioned silver-loaded chitosan coating are derived from silver sulfate.

[0017] According to some of the technical solutions of the present invention, a highly effective anti-infection double-layer artificial skin is provided, wherein the PEG in the above-mentioned Bis-PEG-Acid is PEG6.

[0018] According to some technical solutions of the present invention, a highly effective anti-infection double-layer artificial skin is provided, wherein the pore size of the three-dimensional porous structure in the aforementioned double-layer artificial skin is 100-300 μm. The suitable pore size of the product of the present invention can ensure the ingrowth of surrounding tissues and cells during wound repair. In the present invention, the silver ion layer is loaded in chitosan, and the volume of the chitosan coating per unit area indirectly determines its thickness; the thickness of the cross-linked recombinant humanized collagen dermal matrix material is preferably 1.5-3.5 mm in this solution. If it is too thin, the repair force is weak and it does not meet the requirements for use in full-thickness wounds in clinical practice; if it is too thick, the internal nutrients are difficult to enter, affecting the effect and not conducive to external fixation.

[0019] A preferred embodiment of the present invention is as follows: This invention provides a highly effective anti-infection double-layer artificial skin and its preparation method.

[0020] The double-layer artificial skin has a double-layer structure. The upper layer is a translucent medical polyurethane (with a chitosan coating loaded with silver ions on the inside); the lower layer is a cross-linked recombinant humanized collagen dermal matrix material.

[0021] The preparation method is as follows: 1. Prepare a chitosan-silver sulfate solution with a chitosan concentration of 0.5%-2.5% and a silver content of 1-3% of the chitosan mass. Coat the viscous solution onto a medical polyurethane membrane, applying 0.2-0.5 ml per square centimeter, and then let it stand at 35-45℃ for 2-4 hours to obtain the upper layer structure of the double-layer artificial skin.

[0022] 2. Prepare a recombinant collagen solution (concentration 1-15%), add transglutaminase (mTGase) (0.05–0.2 U / mL), pH 6.0–7.0, and react at 37°C for 1–4 h; then freeze-dry to obtain mTGase-crosslinked recombinant collagen scaffold material; add transglutaminase (0.5-2 μg) per mL of collagen solution (collagen mass 0.01-0.15 g) with an enzyme activity of 100 U / mg.

[0023] 3. Prepare an EDC.HCl (carbodiimide hydrochloride) / Bis-PEG6-Acid (PEG linker) solution (EDC concentration 0.01-1%, PEG linker concentration 0.1-1%). Place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution at a material-to-solution volume ratio of 1:2 for a second crosslinking reaction. The reaction temperature is 25-45℃, and the reaction time is 4-20 hours. After the reaction, wrap the material in a dialysis bag and wash it with purified water in a shaker for 24 hours at a shaker speed of 60-100 rpm. A certain temperature ensures rapid reaction, but it should not be too high, as this will affect the material stability (rigidity) and enzyme activity. The washing time ensures the removal of excess crosslinking reagent. The shaker is used to increase washing efficiency, but it should not be too large, as centrifugal force will affect the spatial structure of the material. Using the method of this invention as a crosslinking agent can minimize the amount of crosslinking agent used while obtaining a skin scaffold material with suitable mechanical strength and tensile strength.

[0024] 4. After cleaning, the material is laid flat on the medical polyurethane membrane prepared in step 1 (the contact side is a chitosan coating loaded with silver ions). After 1 minute, the entire material is placed in a freeze dryer for drying to obtain the final double-layer artificial skin material with high anti-infection properties.

[0025] The present invention has at least the following effects: 1. High cell proliferation and migration effects This material is a recombinant collagen scaffold with a three-dimensional porous structure, which provides cells with a suitable physical structure (100-200μm) to facilitate the adhesion and proliferation of fibroblasts, vascular endothelial cells, etc., and has a significant effect on skin wound repair.

[0026] 2. Significant antibacterial properties The material of this invention has excellent antibacterial effects. Against Gram-positive bacteria, it has an inhibition rate of 99.9% against Staphylococcus aureus (ATCC 6538) and 99.8% against Staphylococcus epidermidis (ATCC 12228), which can effectively reduce the risk of skin and wound infection. Against Gram-negative bacteria, it has an inhibition rate of 99.9% against Escherichia coli (ATCC 25922) and 99.7% against Pseudomonas aeruginosa (ATCC 9027), which can reduce the occurrence of mucosal and instrument-related infections.

[0027] 3. Good modification effect (complementary crosslinking sites) This invention utilizes mTGase to primarily crosslink glutamine (Gln) and lysine (Lys) sites, forming protein-protein crosslinks; Bis-PEG6-Acid (PEG linker) can crosslink Lys-NH2 with another protein, forming PEG-protein crosslinks. Their mechanisms of action differ, and they do not directly synergistically catalyze the same reaction site. Therefore, mTGase first constructs a primary protein network, and Bis-PEG6-Acid further enhances the crosslinking density, achieving a dual-network structure. Ultimately, this enhances the mechanical strength, degradation resistance, and water absorption of the scaffold material.

[0028] The cross-linking method of this invention improves the mechanical strength of the material, and the formation of new chemical bonds also increases its resistance to degradation (including reducing enzymatic hydrolysis sites). The primary structure of recombinant collagen itself has a high proportion of hydrophilic amino acids, and the interconnected porous structure formed by the method of this invention is conducive to water absorption and retention, resulting in excellent degradation resistance and water absorption-related indicators.

[0029] 4. Biocompatibility The cross-linking agents used are highly safe (enzymes and PEG derivatives), and in a double cross-linking form, both are used in small quantities, resulting in materials with good tensile elasticity. They exhibit good biocompatibility and are non-cytotoxic and non-sensitizing. In the cross-linking system of this invention, EDC acts as a catalyst and does not enter the material itself; it can be washed away subsequently. Bis-PEG6-Acid and mTGase have good biocompatibility. Furthermore, Bis-PEG6-Acid has a relatively long molecular length (molecular weight 382.4), and the cross-linked material possesses both mechanical strength and ductility (suitable hardness). Attached Figure Description

[0030] Figure 1 The following are the 1-day effects of different materials used in this embodiment on rat wound repair. Figure 2 The following are the 3-day wound repair effects of different materials used in this embodiment on rats; Figure 3 The following are the 7-day wound repair effects of different materials used in this embodiment on rats; Figure 4 This is a diagram showing the relative wound surface results of rats using different materials in this embodiment; Figure 5 These are scanning electron microscope (SEM) images of different materials used in this embodiment. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more definite definition of the scope of protection of the present invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations. Reagents not specified in the embodiments are all commercially available in the art.

[0032] EDC.HCL: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Bis-PEG6-Acid is the PEG linker and polyethylene glycol linker used in this invention; the raw materials of this invention have better mechanical properties. If the linker is too long, it is more difficult to ensure the pore structure of the material, which affects the cell growth space.

[0033] Example 1: Highly Effective Anti-Infection Bilayer Artificial Skin 1. Prepare a chitosan-silver sulfate solution with a chitosan concentration of 1.25% and a silver content of 2% of the chitosan mass; coat the viscous solution onto a medical polyurethane membrane, applying 0.35 ml per square centimeter, and then let it stand at 40°C for 3 hours to obtain the upper layer structure of the double-layer artificial skin.

[0034] 2. Prepare a recombinant collagen solution (concentration 7.5%), add transglutaminase (mTGase) (1.25 U / mL), pH 6.5, and react at 37°C for 2.5 h; then freeze-dry to obtain mTGase cross-linked recombinant collagen scaffold material; 3. Prepare an EDC (carbodiimide hydrochloride) / Bis-PEG6-Acid (PEG linker) solution (EDC concentration 0.5%, PEG linker concentration 0.5%). Place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution for a second crosslinking reaction at 35℃ for 12 hours. After the reaction, wrap the material in a dialysis bag and wash it with purified water in a shaker for 24 hours at a shaker speed of 80 rpm.

[0035] 4. After cleaning, the material is laid flat on the medical polyurethane membrane prepared in step 1 (the contact side is a chitosan coating loaded with silver ions). After 1 minute, the entire material is placed in a freeze dryer for drying to obtain the final double-layer artificial skin material with high anti-infection properties.

[0036] Example 2: 1. Prepare a chitosan-silver sulfate solution with a chitosan concentration of 0.5% and a silver content of 1% of the chitosan mass; coat the viscous solution onto a medical polyurethane membrane, applying 0.2 ml per square centimeter, and then let it stand at 35°C for 2 hours to obtain the upper layer structure of the double-layer artificial skin.

[0037] 2. Prepare a recombinant collagen solution (concentration 1%), add transglutaminase (mTGase) (0.05 U / mL), pH 6.0, and react at 37°C for 1 h; then freeze-dry to obtain mTGase cross-linked recombinant collagen scaffold material; 3. Prepare an EDC (carbodiimide hydrochloride) / Bis-PEG6-Acid (PEG linker) solution (EDC concentration 0.01%, PEG linker concentration 0.1%). Place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution for a second crosslinking reaction at 25℃ for 4 hours. After the reaction, wrap the material in a dialysis bag and wash it with purified water in a shaker for 24 hours at a shaker speed of 60 rpm.

[0038] 4. After cleaning, the material is laid flat on the medical polyurethane membrane prepared in step 1 (the contact side is a chitosan coating loaded with silver ions). After 1 minute, the material is placed in a freeze dryer for drying to obtain the final double-layer artificial skin material with high anti-infection properties.

[0039] Example 3: 1. Prepare a chitosan-silver sulfate solution with a chitosan concentration of 2.5% and a silver content of 3% of the chitosan mass; coat the viscous solution onto a medical polyurethane membrane, applying 0.5 ml per square centimeter, and then let it stand at 45°C for 4 hours to obtain the upper layer structure of the double-layer artificial skin.

[0040] 2. Prepare a recombinant collagen solution (concentration 15%), add transglutaminase (mTGase) (0.2 U / mL), pH 7.0, and react at 37°C for 4 h; then freeze-dry to obtain mTGase cross-linked recombinant collagen scaffold material; 3. Prepare an EDC (carbodiimide hydrochloride) / Bis-PEG6-Acid (PEG linker) solution (EDC concentration 1%, PEG linker concentration 1%). Place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution for a second crosslinking reaction at 45℃ for 20 hours. After the reaction, wrap the material in a dialysis bag and wash it with purified water in a shaker for 24 hours at a shaker speed of 100 rpm.

[0041] 4. After cleaning, the material is laid flat on the medical polyurethane membrane prepared in step 1 (the contact side is a chitosan coating loaded with silver ions). After 1 minute, the entire material is placed in a freeze dryer for drying to obtain the final double-layer artificial skin material with high anti-infection properties.

[0042] Comparative Example 1 The preparation method is the same as in Example 1, except that the chitosan concentration in step 1 is 0.1% and the silver ion concentration is 0.5%.

[0043] Comparative Example 2 The preparation method is the same as in Example 1, except that the concentration of recombinant collagen solution in step 1 is 0.5%; the amount of transglutaminase (mTGase) added in step 2 is 0.01 U / mL; and the concentration of PEG linker in step 3 is 0.5%.

[0044] Comparative Example 3 The preparation method is the same as in Example 1, except that the silver content in step 1 is 10% of the chitosan mass; and the EDC concentration in step 3 is 2%.

[0045] The following tests will be performed, and the testing methods are as follows: 1. Antibacterial test ① Grouping and Sample Preparation: The sample was divided into a blank group, a control group (commercially available ordinary collagen repair material), and an experimental group (n=3). The material was cut to a diameter of (4.0±0.2) cm². 2 ② Preparation of bacterial suspension: Staphylococcus aureus and Escherichia coli were inoculated into nutrient broth medium and incubated at 37℃ for 18-24 hours to obtain bacterial suspensions in the logarithmic growth phase; Candida albicans was inoculated into Sabouraud dextrose agar and activated by incubation at 37℃ for 24-48 hours. The activated bacterial suspension was then diluted with sterile physiological saline to adjust the bacterial concentration to (1.0 × 10⁻⁶). 5 - 1.0×10 6 ③ Sample inoculation: Take samples from the experimental group and control group, and place them in sterile petri dishes. Add 0.2 mL of the prepared bacterial solution to each sample (for the blank group, add the bacterial solution directly), ensuring that the bacterial solution evenly covers the sample surface. Place the dishes in a biosafety cabinet and let them stand for 30 min to allow the bacterial solution to be fully absorbed by the samples. ④ Shaking elution: Add 10 mL of sterile physiological saline to each petri dish and place them on a shaker to shake and elute (shaking frequency 150 r / min, shaking time 10 min) to fully elute the bacteria adsorbed on the samples into the physiological saline, obtain the eluent, and dilute it 10 times. ⑤ Colony counting: Take 0.1 mL of the elution dilution and evenly add it to the surface of a nutrient agar plate (for fungi, use Sabouraud agar plate). Spread it evenly with a sterile spreader, let it stand for 15 min, and then invert it and incubate it in a 37℃ constant temperature incubator for 24 h (48 h for fungi). After the incubation, count the number of colonies (CFU) on each plate and calculate the average number of colonies for each group.

[0046] The antibacterial rate was calculated based on the average colony counts of the experimental and control groups to quantitatively evaluate the antibacterial effect of the samples. The formula for calculating the antibacterial rate is as follows: Antibacterial rate (%) = (average colony count in blank control group - average colony count in sample group) / average colony count in blank control group × 100%; Evaluation criteria: Antibacterial rate ≥ 90%: the sample has excellent antibacterial properties; 60% ≤ antibacterial rate < 90%: the sample has good antibacterial properties; Antibacterial rate < 60%: the sample has poor antibacterial properties.

[0047] Test result 1:

[0048] As shown in Result 1, the sample prepared by the method in the example has a high antibacterial rate (>90%); in Comparative Example 1, the concentrations of chitosan and silver ions are low, and the antibacterial activity is weaker compared with other groups.

[0049] 2. Mechanical strength testing Referring to the YY / T 0654-2008 (Medical Collagen Sponge) standard, the specimens were cut into rectangular tensile strips. Length (L) = 50 mm, effective working length (i.e., distance between the two clamps, L0) = 20 mm, width (W) = 10 mm. Loading method: axial tension; tensile speed range: 1-5 mm / min (if the sample is brittle, the speed can be reduced to 0.5 mm / min; if it is flexible, it can be appropriately increased to 5 mm / min to ensure a smooth tensile process and accurate data); clamp spacing: 20 mm to ensure the specimen is in a naturally straight state after installation, without pre-tension or relaxation; record the maximum force and elongation at the moment of fracture during the tensile process.

[0050] Test result 2:

[0051] As shown in Result 2, compared with other groups, the sample prepared by the method in Comparative Example 2 has lower mechanical strength and elongation (P<0.05), indicating that the material has lower intermolecular crosslinking.

[0052] 3. Cytotoxicity detection Referring to GB / T 16886.12-2022 Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Samples, exponentially growing L929 cells were digested and collected, and the cell density was adjusted to 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 24 hours of culture in the 96-well plates, the medium was changed, and the sample groups were treated with extraction buffer (3 ml physiological saline / mL). 2Fresh complete culture medium was added to each well for the sample, blank group, and negative control group, while 100 μL of complete culture medium containing 4% DMSO was added to the positive control group. The culture plates were incubated at 37°C in a 5% CO2 incubator for 72 h. After 72 h of incubation, 10 μL of 10 mg / mL MTT solution was added to each well, and the plates were incubated at 37°C in the dark. After 4 h, the supernatant was discarded by centrifugation, and 200 μL of dimethyl sulfoxide was added to each well. The plates were then shaken with a micro-oscillator to fully dissolve the crystals, and the OD value was measured at 490 nm.

[0053] Assess cytotoxicity (the higher the proliferation rate, the lower the cytotoxicity of the material).

[0054] Test result 3:

[0055] As shown in Result 3, compared with other groups, the sample prepared by the method in Comparative Example 3 had a lower relative cell proliferation rate (<80%), indicating that the material has a high cytotoxic effect.

[0056] 4. Wound healing effect Rats were anesthetized and their skin prepared. Intraperitoneal anesthesia was administered using 3% sodium pentobarbital at a dose of 45 mg / kg. After successful anesthesia, the back hair was shaved using electric clippers. A circular, full-thickness skin excision wound of approximately 8 mm in diameter was created on the animal's back using a skin sampler (8 mm in diameter). After model establishment, the wound was covered with the corresponding materials (blank group, control group (commercially available skin repair product [Pinnak] imported Japanese skin repair material, composed of collagen), and experimental group (sample from Example 3)) and secured with tape. Wound size was observed immediately post-surgery (1 day), at 3 days (3 days), and at 7 days (7 days), and statistical analysis was performed. Figures 1 to 3 The effects of different materials on rat wound repair were compared. The control group used commercially available skin repair product [Pinnaike], while the experimental group used samples prepared according to the method in Example 3. * indicates that compared with the experimental group, P<0.05.

[0057] Test result 4: Figure 4 Image showing the relative wound surfaces of rats treated with different materials; As shown in result 4, at 7 days, the experimental group had a significant advantage in wound repair compared with the blank group and the control group (P<0.05).

[0058] 5. Material's resistance to degradation and water absorption Weigh approximately 0.1 g of collagen cross-linking material and add 3 ml of 200 U / ml type I collagenase-physiological buffer solution. Incubate in a 37°C water bath for 24 hours and calculate the degradation rate. Degradation rate = (m before degradation - m after degradation) / m before degradation × 100%.

[0059] The mass of the sample is m1. After being fully hydrated in physiological saline (10s), the mass is m2. The water absorption ratio of each sample is calculated according to the formula: water absorption ratio = (m2-m1) / m1.

[0060] Test result 5:

[0061] Results 5 show that Comparative Example 2 has a high degradation rate and is easily degraded; however, the sample has low water absorption, indicating that the microstructure and mechanical properties of the indirect reaction material are insufficient.

[0062] 6. Scanning electron microscope (SEM) image of the material. The material was taken and longitudinally sectioned, then sputter-coated with gold for 60 seconds. The microstructure was observed using a scanning electron microscope at 200x magnification, and the average pore size was measured using Image Pro Plus software. The SEM structure is shown below. Figure 5 As shown.

[0063] Test result 6:

[0064] Results 6 show that Comparative Example 2 has a low collagen concentration, resulting in collapsed pore structures and uneven pore sizes; Comparative Example 3 has a high cross-linking strength, and the pores formed after freeze-drying are too dense, which is not conducive to the uniform pore size required for cell proliferation.

[0065] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a highly effective anti-infection double-layer artificial skin, characterized in that, The preparation method includes the following steps: Step 1: Prepare a chitosan silver ion solution, coat the chitosan silver ion solution onto an inert polymer film to obtain the upper layer structure of a double-layer artificial skin; Step 2: Prepare a recombinant collagen solution, add mTGase, react, dry, and obtain mTGase-crosslinked recombinant collagen scaffold material; Step 3: Prepare Bis-PEG6-Acid solution, place the mTGase-crosslinked recombinant collagen scaffold material in the prepared EDC / Bis-PEG6-Acid crosslinking solution for a second crosslinking, and wash with purified water after the reaction; Step 4: The cleaned material is laid flat on the inert polymer material film prepared in Step 1, with the contact side being a chitosan coating loaded with silver ions. The entire material is then dried to obtain a highly effective anti-infection double-layer artificial skin.

2. The method for preparing a highly effective anti-infection double-layer artificial skin according to claim 1, characterized in that, In step 1, the inert polymeric material membrane is a polyurethane membrane, silicone, or polyvinyl alcohol. The silver ions are sourced from silver sulfate. The chitosan concentration is 0.5%-2.5%, and the silver content is 1-3% of the chitosan mass. The chitosan silver ion solution is coated onto the inert polymeric material membrane at a rate of 0.2-0.5 ml per square centimeter.

3. The method for preparing a highly effective anti-infection double-layer artificial skin according to claim 2, characterized in that, In step 2, the concentration of the recombinant collagen solution is 1-15%, and 0.05-0.2 U / mL of mTGase is added, with a pH of 6.0-7.

0.

4. The method for preparing a highly effective anti-infection double-layer artificial skin according to claim 3, characterized in that, In step 3, the preparation of Bis-PEG6-Acid solution is the preparation of EDC.HCl / Bis-PEG6-Acid solution.

5. The method for preparing a highly effective anti-infection double-layer artificial skin according to claim 4, characterized in that, In step 3 of the preparation method, an EDC.HCl / Bis-PEG6-Acid solution is prepared, with an EDC concentration of 0.01-1% and a PEG linker concentration of 0.1-1%. The reaction temperature is 25-45℃, and the reaction time is 4-20h. After the reaction, the material is wrapped in a dialysis bag and washed with purified water in a shaker at a speed of 60-100rpm. The drying method in the preparation method is freeze drying.

6. A highly effective anti-infection bilayer artificial skin prepared according to any one of claims 1 to 5.

7. The highly effective anti-infection double-layer artificial skin according to claim 6, characterized in that, The double-layer artificial skin has a double-layer structure comprising an upper layer and a lower layer. The upper layer is a polyurethane membrane, silicone, or polyvinyl alcohol, and the inner side of the upper layer is a chitosan coating loaded with silver ions. The lower layer is a cross-linked recombinant humanized collagen dermal matrix material. The cross-linked recombinant humanized collagen dermal matrix material uses mTGase-crosslinked recombinant collagen scaffold material and is cross-linked with Bis-PEG-Acid. The double-layer artificial skin has a three-dimensional porous structure.

8. The highly effective anti-infection double-layer artificial skin according to claim 7, characterized in that, The silver ions in the chitosan coating loaded with silver ions are derived from silver sulfate.

9. The highly effective anti-infection double-layer artificial skin according to claim 8, characterized in that, The PEG in the Bis-PEG-Acid is PEG6.

10. The highly effective anti-infection double-layer artificial skin according to claim 9, characterized in that, The three-dimensional porous structure of the double-layer artificial skin has a pore size of 100-300μm.

Images