Skin wound repairing material and preparation method thereof

Abstract

The invention relates to a skin wound repairing material and a preparation method thereof, and belongs to the technical field of biological medicines. The repair material disclosed by the invention contains palmitoyl tripeptide-1, methacrylated collagen, cuttlebone extract, recombinant human epidermal growth factor lipidosome, polydopamine nanoparticles, hyaluronic acid, sodium alginate modified silk fibroin and the like in a specific proportion. Wherein the cuttlebone extract is a product which is obtained by carrying out common enzymolysis on cuttlebone through chitinase and nattokinase and then carrying out step-by-step enzymolysis on the cuttlebone through gelatinase and lumbrukinase and is less than 5kDa. The components are prepared into an efficient product by adopting a special method, the stability of the components is improved, the problem that the activity is difficult to maintain is solved, and the skin wound repairing effect is good through reasonable compatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a skin wound repair material and a preparation method thereof. Background Art

[0002] Skin, as the largest and most vital organ in the human body, is the first line of defense against external aggression. From a microscopic perspective, the skin is composed of the epidermis, dermis, and subcutaneous tissue, and each layer works closely together. The stratum corneum in the epidermis can effectively resist physical friction, chemical erosion, and microbial invasion; while the dermis is rich in collagen and elastic fibers, which not only give the skin elasticity and toughness, but also have abundant blood vessels, nerves, and hair follicles, which participate in key physiological processes such as temperature regulation and sensory perception. In terms of immune defense, the defense cells in the skin can promptly identify and capture invading pathogens, initiate immune responses, and protect the body's health.

[0003] However, the skin is directly exposed to the external environment, facing various risks at all times, and is extremely vulnerable to various types of trauma. In accidents, there are burns and scalds caused by high temperatures, and cuts caused by sharp objects; in daily life, there are inadvertent abrasions and lacerations during exercise; long-term bedridden patients suffer from chronic wounds such as pressure sores due to continuous pressure on local tissues and obstructed blood circulation. These traumas are like ruthless demons, not only causing patients excruciating physical pain, but also seriously interfering with their daily lives and reducing their quality of life. If not handled properly, the wound is easily infected by pathogens such as bacteria and fungi, and the inflammation continues to spread, leading to serious complications such as sepsis, posing a huge threat to the patient's life safety.

[0004] With the rapid development of multiple disciplines such as materials science and biomedical engineering, there are many kinds of skin wound repair drugs on the market. For example, natural polymer materials, such as collagen, are an important component of the skin, highly compatible with human tissues, and can provide cells with a natural growth scaffold; chitosan, with its unique antibacterial properties and good biocompatibility, can promote cell adhesion and proliferation; but they also have obvious shortcomings, are easily degraded by enzymatic hydrolysis, and have poor stability, which greatly reduces the repair effect. Bioactive materials such as growth factors can accurately regulate cell proliferation, differentiation and migration, and extracellular matrix components can simulate the microenvironment in the body and significantly accelerate wound healing. However, when used alone, the activity of growth factors is easily affected by factors such as temperature and pH, and it is difficult to maintain long-term activity, which ultimately affects the repair effect. In addition, the repair components of the complex are different, and the unreasonable combination of components will also affect the repair effect. Therefore, it is urgent to develop more stable, efficient and safe skin wound repair materials to provide patients suffering from trauma with better quality healing products and help them recover their health as soon as possible. Summary of the invention

[0005] In view of the problems existing in various existing skin wound repair drugs, such as being vulnerable to degradation, poor stability, difficulty in maintaining activity, etc., and the unreasonable ingredient compatibility, which affects the repair effect. The present invention provides a skin wound repair substance and its preparation method. The designed repair substance contains palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin in specific proportions. Each ingredient is prepared by a special method to obtain a highly efficient product, improving the stability of the ingredients and solving the problem of difficulty in maintaining activity. After reasonable compatibility, it has a good skin wound repair effect. The specific technical solution is as follows: A skin wound repair substance, the repair substance comprises the following raw materials in parts by mass: 8 parts to 12 parts of palmitoyl tripeptide-1, 25 parts to 35 parts of methacrylated collagen, 8 parts to 12 parts of cuttlefish bone extract, 4 parts to 6 parts of recombinant human epidermal growth factor liposome, 5 parts to 10 parts of polydopamine nanoparticles, 5 parts to 10 parts of hyaluronic acid, 10 parts to 15 parts of sodium alginate modified silk fibroin, and the water content is 65wt% to 75wt%; The cuttlefish bone extract contains the product obtained by co-enzymolysis of cuttlefish bone with chitinase and nattokinase for 1h to 1.5h, then enzymolysis with trypsin for 50min to 70min, and then enzymolysis with lumbrokinase for 60min to 80min to obtain components with a molecular weight below 5kDa.

[0006] In the above-mentioned repair substance, the methacrylated collagen includes methacrylated type I collagen and methacrylated type II collagen, and the mass ratio is methacrylated type I collagen: methacrylated type II collagen = (8 - 10):(3 - 5).

[0007] In the above-mentioned repair substance, the preparation method of the cuttlefish bone extract comprises the following steps: crushing cuttlefish bone into powder, adding phosphate buffer solution with a mass 8 times to 12 times that of the cuttlefish bone, adding chitinase and nattokinase, enzymolyzing at 45°C to 55°C for 1h to 1.5h, inactivating the enzyme, adjusting the pH to 8.0 - 8.5, adding trypsin, enzymolyzing at 35°C to 38°C for 50min to 70min, centrifuging, taking the supernatant, adding lumbrokinase, enzymolyzing at 50°C to 55°C for 60min to 80min, ultrafiltering with a 5kDa ultrafiltration membrane to obtain components with a molecular weight below 5kDa, and freeze-drying to obtain the cuttlefish bone extract.

[0008] In the above method for preparing the cuttlebone extract, the phosphate buffer solution is a phosphate buffer solution with a pH of 6 to 7 and a concentration of 0.01 mol / L to 0.015 mol / L; the addition amounts of chitinase and nattokinase are both 1% to 2% of the mass of the cuttlebone; the enzyme inactivation is carried out at 80°C to 90°C for 10 min to 20 min; the addition amount of trypsin is 0.5% to 1% of the mass of the cuttlebone; the centrifugation is carried out at 10000 r / min to 12000 r / min for 20 min to 30 min; the addition amount of lumbrokinase is 0.3% to 0.5% of the mass of the cuttlebone.

[0009] In the above-mentioned repair material, the preparation method of the recombinant human epidermal growth factor liposome includes the following steps: According to the mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = (1 to 3): (100 to 120): (10 to 12): (5 to 6): (1 to 1.5): (0.3 to 0.6): (3 to 5), add distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose to deionized water to prepare a mixed solution, then add recombinant human epidermal growth factor, carry out ultrasonic treatment, and freeze-dry to obtain the recombinant human epidermal growth factor liposome.

[0010] In the above method for preparing the recombinant human epidermal growth factor liposome, the power of the ultrasonic treatment is 200 W to 250 W, and the time of the ultrasonic treatment is 15 min to 20 min.

[0011] In the above-mentioned repair material, the preparation method of the sodium alginate-modified silk fibroin includes the following steps: Prepare an aqueous sodium alginate solution with a mass concentration of 2% to 5%; disperse silk fibroin in water to prepare an emulsion with a mass concentration of 5% to 8%; prepare an aqueous calcium chloride solution with a concentration of 0.1 mol / L to 0.3 mol / L; according to the mass ratio of silk fibroin: sodium alginate: calcium chloride = 1: (0.5 to 1.5): (0.03 to 0.05), under stirring, add the emulsion to the aqueous sodium alginate solution, mix evenly to obtain a mixed solution; under stirring, add the mixed solution to the aqueous calcium chloride solution, stir and crosslink, and freeze-dry to obtain the sodium alginate-modified silk fibroin.

[0012] In the above method for preparing the sodium alginate-modified silk fibroin, the water is all deionized water; the emulsion contains Tween-80, and the content is 1% to 3% of the mass of silk fibroin; the stirring speed when the emulsion is added to the aqueous sodium alginate solution is 100 r / min to 200 r / min; the stirring speed when the mixed solution is added to the aqueous calcium chloride solution is 100 r / min to 200 r / min; the stirring and crosslinking is carried out at 30°C to 40°C and a stirring speed of 100 r / min to 200 r / min for 1 h to 3 h.

[0013] The preparation method of the above-mentioned skin wound repair substance comprises the following steps: by mass parts, add palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin into deionized water, mix them, and adjust them into a paste with a water content of 65wt% - 75wt%, then evacuate and defoam to obtain the repair substance.

[0014] In the above preparation method, the methacrylated collagen is pretreated by swelling and dialysis. Add the methacrylated collagen into deionized water for swelling, then add a dialysis bag with a molecular weight cut-off of 5 kDa, and dialyze in deionized water at 4°C - 6°C for 50h - 60h; the polydopamine nanoparticles are pretreated by dispersion. Add the polydopamine nanoparticles into deionized water and disperse them evenly by ultrasonic treatment; the time for evacuating and defoaming is 15min - 20min.

[0015] A skin wound repair substance and its preparation method provided by the present invention have the following beneficial effects: First, as a signal peptide, palmitoyl tripeptide-1 can stimulate fibroblasts, promote the synthesis of collagen, especially type I and type III collagen, help increase the firmness and elasticity of the skin, reduce the formation of wrinkles, and provide structural support for new tissues during skin wound repair, promoting the reconstruction of the skin at the wound site.

[0016] Second, methacrylated collagen, due to its high affinity with human tissues, can provide a natural growth scaffold for cell adhesion, proliferation, and differentiation. Among them, methacrylated type I collagen is mainly responsible for maintaining the strength and toughness of the skin, and methacrylated type II collagen plays an important role in regulating cell function and tissue repair. When the two are combined in an appropriate mass ratio, it is closer to the natural collagen ratio of the skin and can better simulate the physiological environment of the skin.

[0017] Third, the cuttlefish bone extract is rich in various minerals and bioactive components, has good hemostatic efficacy, can accelerate platelet aggregation, and is mainly used to promote the coagulation process.

[0018] In the present invention, the cuttlefish bone extract is prepared by a multi-step enzymatic hydrolysis process for cuttlefish bone. First, chitinase and nattokinase are used for co-enzymatic hydrolysis to release bioactive small molecule products; then, trypsin and lumbrokinase are used for step-by-step enzymatic hydrolysis to act on specific substrates to obtain more effective specific highly active small peptides and other small molecule active products, fully exerting the biological efficacy. It is found that this specific product can greatly promote cell proliferation and migration in skin repair, accelerate the wound healing speed, and reduce the risk of infection.

[0019] IV. In recombinant human epidermal growth factor liposomes, recombinant human epidermal growth factor can specifically bind to receptors on the cell surface, activate the intracellular signal transduction pathway, regulate cell proliferation, differentiation and migration, and promote the regeneration and repair of epidermal cells; as a carrier, liposomes can protect recombinant human epidermal growth factor from the influence of external environmental factors (such as enzymatic hydrolysis, changes in acidity and alkalinity, etc.), extend its active time, and improve its bioavailability. In particular, the combined protection of dioleoyl phosphatidylethanolamine and phosphatidylserine can further effectively improve the biological stability of recombinant human epidermal growth factor liposomes; this structure can effectively protect the activity of growth factors from being damaged by the external environment and can be stably released in the body, continuously playing a role in promoting cell repair.

[0020] V. Polydopamine nanoparticles have excellent adhesion, can tightly adhere to the wound surface, and enhance the fit between the repair material and the wound; at the same time, their good biocompatibility does not cause obvious immune rejection reactions, and can also promote cell adhesion and growth, which is beneficial to wound healing.

[0021] VI. Hyaluronic acid is a natural moisturizer that can absorb and retain a large amount of water, maintaining a moist environment at the wound site. This helps prevent the wound from drying and crusting excessively, promotes cell migration and proliferation, and can also inhibit inflammatory reactions, providing a good microenvironment for wound healing.

[0022] VII. In sodium alginate-modified silk fibroin, sodium alginate has strong water absorption and can effectively absorb wound exudate, keeping the wound dry; silk fibroin has good biocompatibility and mechanical properties. After the two are combined, sodium alginate-modified silk fibroin with special structures and properties is formed, which combines the advantages of both and shows better performance in wound repair. It can not only provide a physical barrier for the wound but also promote cell adhesion and growth, accelerating wound healing.

[0023] VIII. The specific addition ratios of the components in the present invention are carefully designed. Palmitoyl tripeptide-1 and methacrylated collagen act synergistically to promote the repair and reconstruction of skin tissue at the cellular and molecular levels; cuttlefish bone extract and recombinant human epidermal growth factor liposomes play key roles in promoting growth and cell regulation respectively, accelerating the wound healing process; polydopamine nanoparticles enhance the binding between the repair material and the wound, and hyaluronic acid and sodium alginate-modified silk fibroin jointly maintain the moist and physical barrier functions of the wound. The components cooperate with each other to form an organic whole, exerting a more powerful repair effect than a single component.

[0024] IX. In the preparation method, adding methacrylated collagen to deionized water for swelling is to allow the collagen to fully absorb water, unfold its molecular structure, and expose more active sites, which is beneficial for subsequent interactions with other components. After that, it is placed in a dialysis bag and dialyzed in low-temperature deionized water. On the one hand, it can remove impurities and small-molecule substances and improve the purity of collagen. On the other hand, the low-temperature environment helps to improve the stability of collagen and ensure the integrity of its structure and function.

[0025] X. In the preparation method, ultrasonic pre-dispersion of polydopamine nanoparticles in deionized water is carried out. The cavitation effect of ultrasound can break the aggregation force between polydopamine nanoparticles, making them evenly dispersed in water and forming a stable dispersion system. In this way, during the subsequent preparation of the repair material, the polydopamine nanoparticles can be evenly distributed in the system and fully play their adhesion and cell growth promotion roles.

[0026] In summary, the designed repair material of the present invention contains palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin in specific proportions. Each component is prepared by a special method to obtain a highly efficient product, improving the stability of the components, solving the problem of difficult activity maintenance. After reasonable compatibility, it has a good skin wound repair effect, and the preparation method is simple, with great practical value. Detailed implementation manners

[0027] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0028] Example 1: A skin wound repair material, the repair material includes the following raw materials in parts by mass: 10 parts of palmitoyl tripeptide-1, 30 parts of methacrylated collagen, 10 parts of cuttlefish bone extract, 5 parts of recombinant human epidermal growth factor liposome, 8 parts of polydopamine nanoparticles, 8 parts of hyaluronic acid, 12 parts of sodium alginate-modified silk fibroin, and the water content is 65wt% - 75wt%.

[0029] Among them, the methacrylated collagen includes methacrylated type I collagen and methacrylated type II collagen, and the mass ratio is methacrylated type I collagen: methacrylated type II collagen = 9:4.

[0030] Among them, the preparation method of the cuttlebone extract includes the following steps: The cuttlebone is crushed into powder, 10 times the mass of pH 6.5, 0.012 mol / L phosphate buffer solution is added, 1.5% of chitinase based on the mass of the cuttlebone and 1.5% of nattokinase based on the mass of the cuttlebone are added, enzymolysis is carried out at 50 °C for 1 h, the enzyme is inactivated at 85 °C for 15 min, the pH is adjusted to 8.2, 0.8% of trypsin based on the mass of the cuttlebone is added, enzymolysis is carried out at 37 °C for 60 min, centrifuged at 11000 r / min for 25 min, the supernatant is taken, 0.4% of earthworm kinase based on the mass of the cuttlebone is added, enzymolysis is carried out at 52 °C for 70 min, ultrafiltration is carried out using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, freeze-dried to obtain the cuttlebone extract.

[0031] Among them, the preparation method of the recombinant human epidermal growth factor liposome includes the following steps: By mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 2:110:11:5.5:1.2:0.5:4, distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, ultrasonic treatment is carried out at a power of 220 W for 18 min, and freeze-dried to obtain the recombinant human epidermal growth factor liposome.

[0032] Among them, the preparation method of the sodium alginate modified silk fibroin includes the following steps: Prepare a 3% sodium alginate aqueous solution by mass concentration; Dissolve 2% of Tween-80 based on the mass of the silk fibroin in water, disperse the silk fibroin in water to prepare an emulsion with a mass concentration of 6%; Prepare a 0.2 mol / L calcium chloride aqueous solution; According to the mass ratio of silk fibroin: sodium alginate: calcium chloride = 1:1:0.04, under the stirring state of 150 r / min, add the emulsion to the sodium alginate aqueous solution, mix evenly to obtain a mixed solution; Under the stirring state of 150 r / min, add the mixed solution to the calcium chloride aqueous solution, stir and crosslink at 35 °C and 150 r / min for 2 h, and freeze-dry to obtain the sodium alginate modified silk fibroin. Among them, the water used is all deionized water.

[0033] The preparation method of the above-mentioned skin wound repair material includes the following steps: By mass fraction, methacrylated collagen is added to deionized water for swelling, and then a dialysis bag with a molecular weight cut-off of 5 kDa is added, and dialysis is carried out in deionized water at 4 °C for 55 h, and taken out for standby; The polydopamine nanoparticles are added to 2.5 times the mass of deionized water and ultrasonically dispersed evenly for standby; By mass parts, palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin are added to deionized water, mixed, and adjusted into a paste with a water content of 65wt% to 75wt%. After degassing under vacuum for 18 minutes, a repair product is obtained.

[0034] For convenient detection and comparison, the water content of the repair product sample prepared in this example is controlled at 70wt%.

[0035] Example 2: A skin wound repair product, the repair product comprising the following raw materials in mass parts: 8 parts of palmitoyl tripeptide-1, 25 parts of methacrylated collagen, 8 parts of cuttlefish bone extract, 4 parts of recombinant human epidermal growth factor liposome, 5 parts of polydopamine nanoparticles, 5 parts of hyaluronic acid, 10 parts of sodium alginate modified silk fibroin, with a water content of 65wt% to 75wt%.

[0036] Among them, the methacrylated collagen includes methacrylated type I collagen and methacrylated type II collagen, and the mass ratio is methacrylated type I collagen: methacrylated type II collagen = 8:3.

[0037] Among them, the preparation method of the cuttlefish bone extract includes the following steps: The cuttlefish bone is crushed into powder, 8 times the mass of pH 6, 0.01mol / L phosphate buffer solution is added, 1% of the mass of the cuttlefish bone of chitinase and 1% of the mass of the cuttlefish bone of nattokinase are added, enzymolysis is carried out at 45°C for 1h, the enzyme is inactivated at 80°C for 10min, the pH is adjusted to 8.0, 0.5% of the mass of the cuttlefish bone of trypsin is added, enzymolysis is carried out at 35°C for 50min, centrifuged at 10000r / min for 20min, the supernatant is taken, 0.3% of the mass of the cuttlefish bone of earthworm kinase is added, enzymolysis is carried out at 50°C for 60min, ultrafiltration is carried out with a 5kDa ultrafiltration membrane to obtain components below 5kDa, and freeze-dried to obtain the cuttlefish bone extract.

[0038] Among them, the preparation method of the recombinant human epidermal growth factor liposome includes the following steps: According to the mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 1:100:10:5:1:0.3:3, distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, ultrasonicated at 200W for 15min, and freeze-dried to obtain the recombinant human epidermal growth factor liposome.

[0039] Among them, the preparation method of sodium alginate modified silk fibroin includes the following steps: preparing an aqueous sodium alginate solution with a mass concentration of 2%; dissolving Tween-80 with a mass of 1% of silk fibroin in water, dispersing silk fibroin in water, and preparing an emulsion with a mass concentration of 5%; preparing an aqueous calcium chloride solution with a concentration of 0.1 mol / L; according to the mass ratio of silk fibroin:sodium alginate:calcium chloride = 1:0.5:0.03, adding the emulsion to the aqueous sodium alginate solution under stirring at 100 r / min, mixing evenly to obtain a mixed solution; adding the mixed solution to the aqueous calcium chloride solution under stirring at 100 r / min, stirring and crosslinking at 30 °C and 100 r / min for 1 h, and freeze-drying to obtain sodium alginate modified silk fibroin. Among them, the water used is all deionized water.

[0040] The preparation method of the above-mentioned skin wound repair material includes the following steps: By mass, methacrylated collagen is added to deionized water for swelling, and then a dialysis bag with a molecular weight cut-off of 5 kDa is added, and dialysis is carried out in deionized water at 4 °C for 50 h, and then taken out for standby; polydopamine nanoparticles are added to 2 times the mass of deionized water and ultrasonically dispersed evenly for standby; By mass, palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin are added to deionized water, mixed, and adjusted into a paste with a water content of 65 wt% - 75 wt%, and vacuum degassed for 15 min to obtain a repair material.

[0041] For the convenience of detection and comparison, the water content of the repair material sample prepared in this example is controlled at 70 wt%.

[0042] Example 3: A skin wound repair material, the repair material includes the following raw materials by mass: 12 parts of palmitoyl tripeptide-1, 35 parts of methacrylated collagen, 12 parts of cuttlefish bone extract, 6 parts of recombinant human epidermal growth factor liposome, 10 parts of polydopamine nanoparticles, 10 parts of hyaluronic acid, 15 parts of sodium alginate modified silk fibroin, and the water content is 65 wt% - 75 wt%.

[0043] Among them, methacrylated collagen includes methacrylated type I collagen and methacrylated type II collagen, and the mass ratio is methacrylated type I collagen:methacrylated type II collagen = 10:5.

[0044] Among them, the preparation method of the cuttlebone extract includes the following steps: The cuttlebone is crushed into powder, 12 times the mass of pH 7, 0.015 mol / L phosphate buffer solution is added, 2% of the mass of the cuttlebone of chitinase and 2% of the mass of the cuttlebone of nattokinase are added, enzymolysis is carried out at 55 °C for 1.5 h, enzyme inactivation is carried out at 90 °C for 20 min, the pH is adjusted to 8.5, 1% of the mass of the cuttlebone of trypsin is added, enzymolysis is carried out at 38 °C for 70 min, centrifugation is carried out at 12000 r / min for 30 min, the supernatant is taken, 0.5% of the mass of the cuttlebone of earthworm kinase is added, enzymolysis is carried out at 55 °C for 80 min, ultrafiltration is carried out using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, freeze-dried, and the cuttlebone extract is obtained.

[0045] Among them, the preparation method of the recombinant human epidermal growth factor liposome includes the following steps: By mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 3: 120: 12: 6: 1.5: 0.6: 5, distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, and ultrasonic treatment is carried out at a power of 250 W for 20 min, and freeze-dried to obtain the recombinant human epidermal growth factor liposome.

[0046] Among them, the preparation method of sodium alginate modified silk fibroin includes the following steps: Prepare a 5% sodium alginate aqueous solution by mass concentration; Dissolve 3% of the mass of the silk fibroin of Tween-80 in water, disperse the silk fibroin in water, and prepare an 8% emulsion by mass concentration; Prepare a 0.3 mol / L calcium chloride aqueous solution; According to the mass ratio of silk fibroin: sodium alginate: calcium chloride = 1: 1.5: 0.05, under the stirring state of 200 r / min, add the emulsion to the sodium alginate aqueous solution and mix evenly to obtain a mixed solution; Under the stirring state of 200 r / min, add the mixed solution to the calcium chloride aqueous solution, and stir and crosslink at 40 °C and 200 r / min for 3 h, and freeze-dry to obtain sodium alginate modified silk fibroin. Among them, the water used is all deionized water.

[0047] The preparation method of the above-mentioned skin wound repair material includes the following steps: By mass fraction, add methacrylated collagen to deionized water for swelling, then add a dialysis bag with a cut-off molecular weight of 5 kDa, and dialyze in deionized water at 6 °C for 60 h, and take it out for standby; Add polydopamine nanoparticles to 3 times the mass of deionized water and disperse them evenly by ultrasonic treatment for standby; By mass parts, palmitoyl tripeptide-1, methacryloylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin are added to deionized water and mixed to adjust into a paste with a water content of 65 wt% - 75 wt%. Then, it is vacuum degassed for 20 min to obtain the repair substance.

[0048] For convenient detection and comparison, the water content of the repair substance sample prepared in this example is controlled at 70 wt%.

[0049] Example 4: A skin wound repair substance, the repair substance comprising the following raw materials by mass parts: 8 parts of palmitoyl tripeptide-1, 35 parts of methacryloylated collagen, 8 parts of cuttlefish bone extract, 6 parts of recombinant human epidermal growth factor liposome, 5 parts of polydopamine nanoparticles, 10 parts of hyaluronic acid, 10 parts of sodium alginate modified silk fibroin, with a water content of 65 wt% - 75 wt%.

[0050] Among them, the methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio is methacryloylated type I collagen: methacryloylated type II collagen = 8:5.

[0051] Among them, the preparation method of the cuttlefish bone extract includes the following steps: The cuttlefish bone is crushed into powder, 8 times the mass of pH 6.8, 0.01 mol / L phosphate buffer is added, 2% of the mass of chitinase and 1% of the mass of nattokinase of the cuttlefish bone are added, enzymolysis is carried out at 55 °C for 1 h, enzyme inactivation is carried out at 80 °C for 15 min, the pH is adjusted to 8.1, 0.6% of the mass of the cuttlefish bone of chymotrypsin is added, enzymolysis is carried out at 37 °C for 60 min, centrifuged at 10000 r / min for 20 min, the supernatant is taken, 0.5% of the mass of the cuttlefish bone of earthworm kinase is added, enzymolysis is carried out at 55 °C for 60 min, ultrafiltration is carried out using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, and freeze-dried to obtain the cuttlefish bone extract.

[0052] Among them, the preparation method of the recombinant human epidermal growth factor liposome includes the following steps: By mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 3:100:12:5:1.5:0.3:5. Di-stearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, ultrasonicated at 200 W for 15 min, and freeze-dried to obtain the recombinant human epidermal growth factor liposome.

[0053] Among them, the preparation method of sodium alginate modified silk fibroin includes the following steps: preparing an aqueous solution of sodium alginate with a mass concentration of 5%; dissolving Tween-80 accounting for 1.5% of the mass of silk fibroin in water, dispersing silk fibroin in water, and preparing an emulsion with a mass concentration of 7%; preparing an aqueous calcium chloride solution with a concentration of 0.3 mol / L; according to the mass ratio of silk fibroin:sodium alginate:calcium chloride = 1:1.2:0.03, adding the emulsion to the aqueous sodium alginate solution under stirring at 200 r / min, mixing evenly to obtain a mixed solution; adding the mixed solution to the aqueous calcium chloride solution under stirring at 200 r / min, stirring and crosslinking at 37 °C and 200 r / min for 2 h, and freeze-drying to obtain sodium alginate modified silk fibroin. Among them, the water used is all deionized water.

[0054] The preparation method of the above-mentioned skin wound repair material includes the following steps: By mass, add methacrylated collagen to deionized water for swelling, then add a dialysis bag with a molecular weight cut-off of 5 kDa, and dialyze in deionized water at 4 °C for 52 h, and take it out for standby; add polydopamine nanoparticles to 2 times the mass of deionized water and disperse them evenly by ultrasonic treatment for standby; By mass, add palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin to deionized water, mix them, and adjust them into a paste with a water content of 65 wt% to 75 wt%, and evacuate and defoam for 15 min to obtain a repair material.

[0055] For the convenience of detection and comparison, the water content of the repair material sample prepared in this example is controlled at 70 wt%.

[0056] In the above embodiments: Palmitoyl tripeptide-1 is sourced from Shaanxi Haibo Biotechnology Co., Ltd. Methacrylated type I collagen and methacrylated type II collagen are sourced from Shanghai Jiadel Chemical Technology Co., Ltd. Sepia officinalis is sourced from Bozhou Yuanshengtang Pharmaceutical Co., Ltd., cuttlefish bone. Recombinant human epidermal growth factor (EGF) is sourced from Guangzhou Qibao Biotechnology Co., Ltd. Polydopamine nanoparticles are sourced from Beijing Zhongke Keyou Technology Co., Ltd. Hyaluronic acid is sourced from Lanli Biotechnology (Xi'an) Co., Ltd., model LL-TMZS. Sodium alginate is sourced from Qiyuan (Guangdong) Pharmaceutical and Chemical Co., Ltd., with a purity of over 99%. Silk fibroin is sourced from Xi'an Dongchi Biotechnology Co., Ltd., silk fibroin, a molecular fibrous protein extracted from silk. Chitinase is sourced from Guangdong Jiudian Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Nattokinase is sourced from Shaanxi Langde Biotechnology Co., Ltd., with an enzyme activity of 40,000 U / g. Trypsin is sourced from Shanghai Yiji Industrial Co., Ltd., α-chymotrypsin, model M0002, with an enzyme activity of 1500 U / mg. Lumbrokinase is sourced from Tianben Biotechnology (Shanxi) Co., Ltd., with an enzyme activity of 10,000 iu / mg. Phosphatidylcholine is sourced from Shanxi Hengtian Biotechnology Co., Ltd., egg yolk lecithin. Dioleoyl phosphatidylethanolamine (DOPE) is sourced from Shaoguan Chengfeng Chemical Industry Co., Ltd. Cholesterol is sourced from Jiangsu Yungao Biotechnology Co., Ltd., pharmaceutical grade. Phosphatidylserine is sourced from Shaanxi Longzhou Biotechnology Co., Ltd., extracted from soybeans. Tween-80 is sourced from Shandong Taixi Chemical Industry Co., Ltd.

[0057] Comparative Example 1 All methacrylated collagen is methacrylated type I collagen; other parameters and methods are the same as in Example 1.

[0058] Comparative Example 2 All methacrylated collagen is methacrylated type II collagen; other parameters and methods are the same as in Example 1.

[0059] Comparative Example 3 The Sepia officinalis extract is replaced with Sepia officinalis powder; other parameters and methods are the same as in Example 1.

[0060] Comparative Example 4 In the preparation method of the Sepia officinalis extract, chitinase and nattokinase are not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0061] Comparative Example 5 In the preparation method of the Sepia officinalis extract, trypsin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0062] Comparative Example 6 In the preparation method of the Sepia officinalis extract, lumbrokinase is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0063] Comparative Example 7 Recombinant human epidermal growth factor was not prepared into liposomes, and recombinant human epidermal growth factor was directly added in proportion; other parameters and methods were the same as those in Example 1.

[0064] Comparative Example 8 In the preparation method of recombinant human epidermal growth factor liposomes, dioleoyl phosphatidylethanolamine and phosphatidylserine were not added; other parameters and methods were the same as those in Example 1.

[0065] Comparative Example 9 Silk fibroin was not modified with sodium alginate, and silk fibroin was directly added in proportion; other parameters and methods were the same as those in Example 1.

[0066] Comparative Example 10 4 parts (too little) of Sepia extract and 16 parts (too much) of palmitoyl tripeptide-1 were added; other parameters and methods were the same as those in Example 1.

[0067] Comparative Example 11 12 parts (too little) of methacrylated collagen and 30 parts (too much) of sodium alginate-modified silk fibroin were added; other parameters and methods were the same as those in Example 1.

[0068] I. Detection of cell proliferation rate: The MTT colorimetric method was used: Fibroblasts were pre-cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a 37°C, 5% CO2 incubator. During the experiment, the fibroblasts in the logarithmic growth phase were digested with trypsin and seeded into a 96-well plate at a density of 5000 cells / well, with a volume of 200 μL per well. After being placed in the incubator for 24 h and the cells adhered, the old medium was carefully aspirated. The skin wound repair materials of different examples and comparative examples were diluted 10 times in mass with the above DMEM medium to obtain a repair material solution, and 100 μL of the repair material solution was added to each well, with 5 replicates in each group. At the same time, a control group was set up, and only 100 μL of DMEM medium was added to the control group. After continued culture for 48 h, 20 μL of MTT solution (mass concentration: 5 mg / mL, prepared with PBS buffer) was added to each well, and incubated in the 37°C incubator in the dark for 4 h. After the incubation was completed, the supernatant was carefully aspirated, and 150 μL of DMSO was added to each well, and shaken on a shaker at low speed for 10 min to fully dissolve the crystals. Finally, the absorbance value (OD value) at 490 nm was measured on an enzyme-linked immunosorbent assay reader. The cell proliferation rate = (OD value of the experimental group - OD value of the control group) / OD value of the control group × 100%. The average detection results are shown in Table 1 below.

[0069] Table 1 Detection results of cell proliferation rate

[0070] As can be seen from the above results, the prostheses of Examples 1 to 4 can more efficiently promote cell proliferation. In Comparative Example 1 and Comparative Example 2, a single type of methacrylated collagen cannot provide a comprehensive and suitable growth microenvironment for cells like the two collagens mixed in a specific ratio in the examples; the structure and composition of collagen affect cell adhesion, spreading, and proliferation signal transduction. A single collagen weakens the interaction between cells and the material, thus inhibiting cell proliferation. In Comparative Example 3, the cuttlebone powder was not enzymatically treated, and its internal active ingredients such as polysaccharides and polypeptides were difficult to release, unable to effectively stimulate cell proliferation activities. In Comparative Examples 4 to 6, enzymatic hydrolysis was lacking in different stages during the preparation of the cuttlebone extract, resulting in incomplete extracted components, especially the lack of specific small-molecule active peptides, which disrupted the synergistic mechanism for promoting cell proliferation in the cuttlebone extract and decreased its ability to promote cell proliferation. In Comparative Example 7, recombinant human epidermal growth factor was not prepared into liposomes and was easily inactivated by factors such as proteases, temperature, and pH in solution, unable to effectively bind to cell surface receptors and initiate the intracellular proliferation signaling pathway, so the cell proliferation rate was low. In Comparative Example 8, dioleoyl phosphatidylethanolamine and phosphatidylserine were lacking during the preparation of recombinant human epidermal growth factor liposomes. These two components are crucial for maintaining the stability of the bilayer membrane structure of liposomes and promoting the fusion of liposomes with cell membranes. The lack of them would lead to unstable liposome structures, and recombinant human epidermal growth factor would be more likely to leak and inactivate, affecting cell proliferation. In Comparative Example 9, the surface chemical groups and structure of unmodified silk fibroin are not conducive to cell adhesion and growth signal transmission. Compared with the sodium alginate-modified silk fibroin after modification, its compatibility with cells is poor and not conducive to cell proliferation. In Comparative Examples 10 and 11, the component ratios were changed, breaking the optimal synergistic ratio relationship among the components and affecting the promotion of cell proliferation. Excessive or insufficient amounts of certain components would interfere with cell uptake of nutrients, signal transduction, and the construction of the extracellular matrix, thus reducing the cell proliferation rate.

[0071] II. Detection of wound healing time: Healthy adult SD rats weighing 200 g - 250 g were selected, adaptively fed for one week, and allowed free access to food and water. One day before the experiment, the back of the rats was depilated in an area of approximately 5 cm × 5 cm to avoid skin damage. After depilation, the rats were randomly divided into different groups with 5 rats in each group. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (at a dose of 30 mg / kg). After the rats were anesthetized, two full-thickness skin defect wounds with a diameter of 8 mm were made symmetrically on both sides of the spinal column on the back of each rat. After the wound making was completed, the wounds were rinsed with sterile normal saline to remove blood and tissue debris. Different examples and comparative examples of skin wound repair materials were applied to the wounds with a thickness of approximately 1 mm, and then the wounds were covered with sterile gauze and fixed with medical tape. The gauze was changed every day, and the wound healing condition was observed. The key time points such as scab formation, scab shedding, and complete epithelialization of the wounds were recorded. The wound healing time was recorded with the complete epithelialization of the wound and the formation of a complete scar as the healing standard. The test results are shown in Table 2 below.

[0072] Table 2 Test Results of Wound Healing Time As can be seen from the above results, the restorations in Examples 1 to 4 can provide continuous, stable and good bioactivity after contacting the wound, continuously promote the repair of the skin, improve the repair effect and shorten the repair time. In Comparative Examples 1 and 2, the improper proportion of methacrylated collagen affected the cell adhesion and proliferation processes; the appropriate proportion of methacrylated type I collagen and type II collagen could synergistically promote the migration, proliferation of fibroblasts and the synthesis of extracellular matrix; single or unbalanced collagen would lead to abnormal interaction between cells and materials and delay the wound healing process. In Comparative Example 3, the active ingredients of cuttlefish bone powder were not fully released, lacking small molecular substances with specific activities and specific small molecular peptides for promoting repair, and could not effectively promote cell proliferation, angiogenesis and inflammation regression, which was not conducive to wound healing. In Comparative Examples 4 to 6, incomplete enzymatic hydrolysis resulted in incomplete active ingredients for promoting healing in the cuttlefish bone extract, affecting its ability to promote wound healing. Chitinase, nattokinase, trypsin and lumbrokinase played a key role in the degradation of cuttlefish bone components and the release of active ingredients at different stages. The lack of specific enzymatic hydrolysis components would reduce their efficacy. In Comparative Example 7, recombinant human epidermal growth factor was unstable and easily inactivated, and could not effectively promote cell proliferation and migration, especially the migration and coverage of epithelial cells on the wound surface, resulting in an extended wound healing time. In Comparative Example 8, the structural defect of liposome affected the activity of recombinant human epidermal growth factor, making it unable to play its normal role in promoting wound healing; dioleoyl phosphatidylethanolamine and phosphatidylserine were crucial for maintaining the liposome structure and protecting the activity of growth factor, and their absence would reduce the effectiveness of growth factor. In Comparative Example 9, unmodified silk fibroin was not conducive to cell migration and proliferation, and could not provide a good growth microenvironment for cells like sodium alginate-modified silk fibroin, delaying wound healing. In Comparative Examples 10 and 11, the unbalanced component ratio affected the overall repair effect. The proportional relationship among various components had an important impact on cell behavior, inflammatory response and tissue remodeling during the wound healing process. The change in proportion would disrupt the repair balance and lead to an extended healing time.

Claims

1. A skin wound repair material, characterized in that The prosthesis comprises raw materials in the following parts by mass: 8 to 12 parts of palmitoyl tripeptide-1, 25 to 35 parts of methacrylated collagen, 8 to 12 parts of cuttlefish bone extract, 4 to 6 parts of recombinant human epidermal growth factor liposome, 5 to 10 parts of polydopamine nanoparticles, 5 to 10 parts of hyaluronic acid, 10 to 15 parts of sodium alginate modified silk fibroin, with a water content of 65wt% to 75wt%; The cuttlefish bone extract contains the product of components below 5kDa obtained by co-enzyme hydrolysis of cuttlefish bone with chitinase and nattokinase for 1h to 1.5h, followed by enzyme hydrolysis with trypsin for 50min to 70min, and then enzyme hydrolysis with lumbrokinase for 60min to 80min.

2. The skin wound repair product according to claim 1, wherein The methacrylated collagen includes methacrylated type I collagen and methacrylated type II collagen, and the mass ratio is methacrylated type I collagen: methacrylated type II collagen = (8 - 10):(3 - 5).

3. A skin wound repair product according to claim 1, wherein, The preparation method of the cuttlefish bone extract comprises the following steps: crushing cuttlefish bone into powder, adding phosphate buffer solution with a mass 8 to 12 times that of cuttlefish bone, adding chitinase and nattokinase, carrying out enzyme hydrolysis at 45°C to 55°C for 1h to 1.5h, inactivating the enzyme, adjusting the pH to 8.0 to 8.5, adding trypsin, carrying out enzyme hydrolysis at 35°C to 38°C for 50min to 70min, centrifuging, taking the supernatant, adding lumbrokinase, carrying out enzyme hydrolysis at 50°C to 55°C for 60min to 80min, carrying out ultrafiltration with a 5kDa ultrafiltration membrane to obtain components below 5kDa, and freeze-drying to obtain the cuttlefish bone extract.

4. The skin wound repair product according to claim 3, wherein The phosphate buffer solution is a phosphate buffer solution with pH 6 to 7 and 0.01mol / L to 0.015mol / L; the addition amounts of both chitinase and nattokinase are 1% to 2% of the mass of cuttlefish bone; the enzyme inactivation is carried out at 80°C to 90°C for 10min to 20min; the addition amount of trypsin is 0.5% to 1% of the mass of cuttlefish bone; the centrifugation is carried out at 10000r / min to 12000r / min for 20min to 30min; the addition amount of lumbrokinase is 0.3% to 0.5% of the mass of cuttlefish bone.

5. A skin wound repair material according to claim 1, characterized in that, The preparation method of the recombinant human epidermal growth factor liposome comprises the following steps: according to the mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoyl phosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = (1 - 3):(100 - 120):(10 - 12):(5 - 6):(1 - 1.5):(0.3 - 0.6):(3 - 5), adding distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, cholesterol, phosphatidylserine and glucose into deionized water to prepare a mixed solution, then adding recombinant human epidermal growth factor, carrying out ultrasonic treatment, and freeze-drying to obtain the recombinant human epidermal growth factor liposome.

6. The skin wound repair article according to claim 5, characterized in that, The power of the ultrasonic treatment is 200W to 250W, and the time of the ultrasonic treatment is 15min to 20min.

7. A skin wound repair product according to claim 1, characterized in that, The preparation method of sodium alginate modified silk fibroin comprises the following steps: preparing an aqueous sodium alginate solution with a mass concentration of 2% - 5%; dispersing silk fibroin in water to prepare an emulsion with a mass concentration of 5% - 8%; preparing an aqueous calcium chloride solution with a concentration of 0.1 mol / L - 0.3 mol / L; according to the mass ratio of silk fibroin:sodium alginate:calcium chloride = 1:(0.5 - 1.5):(0.03 - 0.05), adding the emulsion into the aqueous sodium alginate solution under stirring, mixing evenly to obtain a mixed solution; adding the mixed solution into the aqueous calcium chloride solution under stirring, stirring and crosslinking, and freeze-drying to obtain sodium alginate modified silk fibroin.

8. A skin wound repair product according to claim 7, characterized in that, The water is all deionized water; the emulsion contains Tween-80, and the content is 1% - 3% of the mass of silk fibroin; the stirring speed when the emulsion is added into the aqueous sodium alginate solution is 100 r / min - 200 r / min; the stirring speed when the mixed solution is added into the aqueous calcium chloride solution is 100 r / min - 200 r / min; the stirring and crosslinking is carried out at 30°C - 40°C and a stirring speed of 100 r / min - 200 r / min for 1 h - 3 h.

9. The preparation method of a skin wound repair product according to claim 1, characterized in that, The preparation method comprises the following steps: according to the mass parts, adding palmitoyl tripeptide-1, methacrylated collagen, cuttlefish bone extract, recombinant human epidermal growth factor liposome, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin into deionized water, mixing, and adjusting to a paste with a water content of 65 wt% - 75 wt%, and performing vacuum defoaming to obtain a repair product.

10. The preparation method of a skin wound repair material according to claim 9, characterized in that, The methacrylated collagen is subjected to swelling and dialysis pretreatment. The methacrylated collagen is added into deionized water for swelling, and then a dialysis bag with a molecular weight cut-off of 5 kDa is added, and dialysis is carried out in deionized water at 4°C - 6°C for 50 h - 60 h; the polydopamine nanoparticles are subjected to dispersion pretreatment, and the polydopamine nanoparticles are added into deionized water and ultrasonically dispersed evenly; the time for vacuum defoaming is 15 min - 20 min.

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