Electrostatic field based electrohydrodynamic jetting of jellyfish collagen hydrogel dressing and method of making the same

Abstract

The application discloses a kind of based on electrostatic field electrofluid jet jellyfish collagen protein hydrogel dressings and preparation method thereof, comprising the following steps: (1) after jellyfish raw material is pretreated, collagen protein is extracted using ultrasonic-assisted acid-enzyme complex, and jellyfish collagen protein is prepared;(2) by ultraviolet activation cage collagen mimic peptide CMP is combined with jellyfish collagen protein, and collagen protein hybrid peptide is prepared;(3) by high pressure microjet homogenizer, jellyfish collagen protein, collagen protein hybrid peptide and hyaluronic acid are uniformly mixed, and multi-component protein composite system is prepared, as natural biological ink;(4) under the condition of sterile vacuum, the water jellyfish collagen protein hydrogel dressings is prepared by electrostatic field electrofluid jet 3D printing.The application uses natural marine extract, while giving consideration to high hygroscopicity, high permeability and biocompatibility at low sensitization, realizes antibacterial hemostatic performance and promotes the growth of new tissue.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a hydrogel dressing for jellyfish collagen based on electrostatic field current fluid spraying and its preparation method. Background Technology

[0002] Collagen, a structural protein found primarily in animal skin, cartilage, tendons, ligaments, and other connective tissues, contains rare amino acids such as hydroxyproline, proline, and hydroxylysine, which are largely absent in other proteins. Due to its advantages in low immunogenicity, coordination with host cells and tissues, hemostatic effects, biodegradability, and physical and mechanical properties, it has become an ideal biomaterial for applications in medicine, clinical medicine, and clinical treatment.

[0003] For large-area skin defects, especially those that are difficult for the body to heal on their own, it is generally necessary to use autologous skin or skin repair materials for auxiliary repair. During wound repair, collagen, the most basic and essential structural macromolecule of the extracellular matrix, plays a crucial role in its synthesis, metabolism, and breakdown. Type I collagen, in particular, as an important type of collagen in the skin structure and closely related to skin tension, plays a particularly important role.

[0004] Currently, some commercially available collagen sources are mainly the skin or tendon tissues of mammals such as pigs and cattle; or recombinant collagen. Recombinant collagen refers to collagen produced by using recombinant DNA technology to genetically manipulate and / or modify the gene encoding the desired human collagen, then using plasmids or viral vectors to deliver the target gene into appropriate host cells, where it is expressed and translated into a collagen polypeptide. This polypeptide is then extracted and purified, offering advantages such as low immunogenicity and good uniformity. However, compared to natural macromolecular collagen, there is no evidence that it possesses a stable triple-helix structure similar to natural collagen, resistant to proteases. Currently, more than 30 proteins have been confirmed to interact with natural collagen, but only a few can interact with recombinant collagen, raising questions about its biological activity. Therefore, utilizing abundant and low-cost marine organisms as a collagen source to replace terrestrial mammals to produce repair materials with weaker antigenicity, better tissue compatibility, and higher biological activity has broad commercial value.

[0005] The existing collagen dressings have the following problems: (1) Traditional gauze dressings have limitations in adsorption capacity and hemostatic efficacy for wound exudate in clinical applications, and have poor breathability; (2) Most dressings on the market currently achieve their functional effects through chemical raw materials and terrestrial collagen, which often cause allergies when used, and in some cases, cause skin scars that are difficult to repair; (3) The thermal denaturation temperature of aquatic collagen is generally lower than 30°C, and its thermal stability is worse than that of mammalian collagen; (4) Current hydrogel dressings still face the problems of low mechanical strength, poor adhesion, and multiple replacements and limited applicability during use. In response to the drawbacks of low extraction rate and easy denaturation of jellyfish collagen, CN101918047B and CN110713534A respectively disclosed a colloidal collagen gel made from jellyfish and a collagen peptide with photoaging improvement effect and its preparation method. However, the patents still have the following problems: (1) the extracted collagen is a small molecular weight peptide, and its structural and activity integrity cannot be guaranteed; (2) there is a lack of optimization of collagen purity.

[0006] In view of the limited ability of the above gauze dressings to absorb exudate and stop bleeding, as well as the disadvantage of sensitization, CN115770323B discloses a recombinant collagen gel dressing and its preparation method and application. However, this patent still has the following problems: (1) The dense structure of the material leads to low gas exchange efficiency; (2) The addition of various chemical reagents such as thickeners and stabilizers to enhance its mechanical strength has a certain impact on wound healing; (3) It is limited by the location and shape of the wound.

[0007] In response to the above-mentioned drawbacks of the current hydrogel dressings, such as poor air permeability and adhesion and limitations due to the location of the wound during use, CN105031713A discloses a 3D bioprinted medical dressing and its preparation method. However, the following problems still exist: (1) The three-dimensional porous structure is used during printing, which has good air permeability but low mechanical strength; (2) The printing temperature and platform temperature are too high, and the collagen activity cannot be guaranteed during the preparation process. Summary of the Invention

[0008] To address the aforementioned problems in existing technologies, this invention provides a jellyfish collagen hydrogel dressing based on electrostatic field electrofluid jetting and its preparation method. This invention utilizes natural marine extracts, achieving low allergenicity while maintaining high hygroscopicity, high permeability, and biocompatibility, thus realizing antibacterial and hemostatic properties as well as promoting new tissue growth.

[0009] The technical solution of the present invention is as follows: The first objective of this invention is to provide a method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field current jet spraying, comprising the following steps: (1) After pretreatment of jellyfish raw materials, collagen was extracted by ultrasound-assisted acid-enzyme complex extraction to obtain jellyfish collagen; (2) Collagen hybrid peptides were prepared by activating cage-like collagen mimic peptide CMP with ultraviolet light and binding it to jellyfish collagen. (3) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid are uniformly mixed by a high-pressure microfluidic homogenizer to prepare a multi-component protein complex system as a natural bio-ink. (4) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing.

[0010] In one embodiment of the present invention, in step (1), the method for pretreating jellyfish raw material is as follows: the jellyfish raw material is mixed with an aqueous solution of sodium hydroxide, stirred for 1-3 days, centrifuged, and the precipitate is washed with deionized water until neutral to obtain pretreated jellyfish raw material.

[0011] In one embodiment of the present invention, in step (1), the concentration of the sodium hydroxide aqueous solution is 0.1 mol / L; the mass-to-volume ratio of the jellyfish raw material to the sodium hydroxide aqueous solution is 1:10-30 g / mL.

[0012] In one embodiment of the present invention, in step (1), the method of ultrasonic-assisted acid-enzyme complex extraction of collagen is as follows: pretreated jellyfish raw material is mixed with acetic acid aqueous solution, pepsin is added and ultrasonic extraction is performed for 2-6 hours; freeze drying is performed to obtain jellyfish collagen.

[0013] In one embodiment of the present invention, the concentration of the aqueous acetic acid solution is 0.5 mol / L.

[0014] In one embodiment of the present invention, the frequency of the ultrasound is 60-70 kHz and the duration of the ultrasound is 60-100 min.

[0015] In one embodiment of the present invention, the mass-to-volume ratio of the pretreated jellyfish raw material to the acetic acid aqueous solution is 1:1-2 g / mL.

[0016] In one embodiment of the present invention, the amount of pepsin used is 1-3% of the mass of the pretreated jellyfish raw material.

[0017] In one embodiment of the present invention, in step (2), the jellyfish collagen obtained in step (1) is dissolved in water to form a jellyfish gel protein solution of 3.5-4 mg / mL; the jellyfish gel protein solution is mixed with CMP aqueous solution, then activated by ultraviolet light, and incubated at 4 °C for 2-4 h.

[0018] In one embodiment of the present invention, in step (2), the concentration of the CMP aqueous solution is 1-1.5 μM / μL; the volume ratio of the CMP aqueous solution to the jellyfish gel protein solution is 1:5-10.

[0019] In one embodiment of the present invention, in step (2), ultraviolet activation uses 365 nm UV light with an intensity of 10 mW / cm². 2 .

[0020] In one embodiment of the present invention, in step (3), the parameters of the high-pressure micro-jet homogenizer are: ultrasonic-assisted processing conditions are ultrasonic power 400-500 W, ultrasonic time 10-20 min, micro-jet pressure 75-125 MPa, and micro-jet 6-9 times.

[0021] In one embodiment of the present invention, in step (3), the mass ratio of jellyfish collagen, collagen hybrid peptide and hyaluronic acid is 5-15:1-2:20-40.

[0022] In one embodiment of the present invention, in step (4), the electrostatic field current fluid jet (EHDP) operating voltage is 1300-1500 V, the working distance is 1.5-2 mm, the ink flow rate is 1-7 μL / min, and the receiving substrate speed is 20-40 mm / s.

[0023] In one embodiment of the present invention, in step (4), the nozzle is a multi-nozzle array nanoscale nozzle with a nozzle size of 200-250 nm.

[0024] The second objective of this invention is to provide a hydrogel dressing of jellyfish collagen based on electrostatic field jet spraying, prepared by the above-described method.

[0025] The beneficial technical effects of this invention are as follows: This invention utilizes microwave-assisted acid-enzyme combined extraction. Ultrasonic treatment induces structural and functional changes in proteins, including reduction in particle size, modification of secondary protein structures, and alterations in solubility. Ultrasound generates significant shear force and mechanical energy, leading to cavitation, surface peeling, erosion, and particle breakage. This improves substrate accessibility to the enzyme and collagen extraction rate, while effectively reducing extraction time.

[0026] This invention utilizes photo-triggered binding of CMP to jellyfish collagen to form a triple-helix hybrid collagen peptide with type I collagen (jellyfish collagen). This peptide exhibits highly specific binding ability against matrix metalloproteinases (MMPs), inhibiting MMP activity and thereby regulating the degradation process of ECM, promoting normal wound healing. This invention employs high-pressure microfluidic homogenization technology to blend marine-derived collagen, recombinant collagen-binding peptides, and hydrophilic polysaccharides, effectively improving the thermosensitivity defects of traditional marine collagen. This composite system exhibits significantly better thermal stability than single-component systems while maintaining bioactivity.

[0027] This invention achieves an ideal balance between mechanical support and gas diffusion efficiency in the fabricated irregular truss structure using electrostatic hydrofluidic jet 3D printing technology. It maintains appropriate mechanical properties to ensure integrity while possessing suitable porosity for oxygen permeability. Notably, the entire processing completely avoids high-temperature treatment, thus ensuring the structural integrity of the collagen and its active ingredients in the dressing solution. Collagen hybrid peptides are embedded in the truss structure; these bioactive units specifically recognize collagen breaks in the damaged area, achieving synergistic bioactivity in wound microenvironment regulation and tissue regeneration while maintaining scaffold structural stability.

[0028] The jellyfish collagen dressing obtained by this invention can be customized according to the location and shape of the wound, and can meet the needs of wounds in different locations on the body. Attached Figure Description

[0029] Figure 1 The extraction rates of jellyfish collagen in Example 1 and Comparative Example 1 of this invention; Figure 2 The images show gel electrophoresis diagrams of jellyfish collagen extracted in Example 1 and Comparative Example 2 of this invention.

[0030] Figure 3 This is a schematic diagram of the irregular truss structure of the electrostatic field current fluid jet jellyfish collagen dressing of the present invention.

[0031] Figure 4 This is a comparison diagram of the in vitro cytotoxicity experiments of the jellyfish collagen hydrogel dressings of Examples 1-4 and Comparative Examples 3-5 of the present invention.

[0032] Figure 5 The results of the jellyfish collagen hydrogel dressings of Examples 1-4 and Comparative Examples 3-5 on the healing of skin wounds in rats are presented. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0035] Figure 3 This is a schematic diagram of the irregular truss structure of the electrostatic field current-jellyfish collagen dressing based on the present invention. The structure is square. The spherical nodes are inlaid with collagen hybrid peptides, and the six-way support truss material is a multi-component protein composite system.

[0036] Cage-shaped collagen mimic peptide CMP was purchased from Xi'an Tianfeng Biotechnology Co., Ltd.; other raw materials are commercially available unless otherwise specified.

[0037] Example 1 A method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field current fluid jetting includes the following steps: (1) Pre-treated jellyfish raw material: soaked in deionized water 3 times, 3 hours each time, and pulped; add 20 mL of 0.1 mol / L sodium hydroxide aqueous solution for every 1 g of jellyfish, soak for 1 day, and wash with water until neutral to obtain pre-treated jellyfish raw material; (2) Take the pretreated jellyfish raw material, add 1.5 mL of 0.5 mol / L acetic acid aqueous solution for every 1 g of pretreated jellyfish raw material, add 3% pepsin, sonicate at a frequency of 65 KHz for 80 min, extract at 4℃ for 4 h, centrifuge, take the supernatant to obtain crude collagen, dialyze in 0.02 mol / L Na2HPO4 solution for 24 h, freeze dry to obtain jellyfish collagen; (3) Dissolve the jellyfish collagen obtained in step (2) in water to form a jellyfish gel protein solution of 3.7 mg / mL; mix the jellyfish gel protein solution with CMP aqueous solution (1.2 μM / μL) at a volume ratio of 1:8, and then use 365 nm UV light (light intensity 10 mW / cm²) to test the mixture. 2 Activation, incubation at 4 ℃ for 3 h, yielding collagen hybrid peptides; (4) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid were homogenized in a mass ratio of 15:1:30 using a microfluidic homogenizer. The ultrasonic-assisted treatment conditions were 450 W ultrasonic power, 15 min ultrasonic time, 100 MPa microfluidic pressure and 7 microfluidic cycles to prepare a multi-component protein complex, which was used as a natural bio-ink. (5) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing. The printhead used is a multi-nozzle array nanoscale nozzle with a nozzle size of 218 nm, an operating voltage of 1400 V applied to the nozzle, an operating distance of 1.8 mm, an ink flow rate of 1 μL / min, and a substrate receiving speed of 30 mm / s.

[0038] Example 2 A method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field current fluid jetting includes the following steps: (1) Pre-treated jellyfish raw material: soaked in deionized water 3 times, 2.5 h each time, and pulped; add 25 mL of 0.1 mol / L sodium hydroxide aqueous solution per 1 g of jellyfish, soak for 2 days, wash with water until neutral, and obtain pre-treated jellyfish raw material; (2) Take the pretreated jellyfish raw material, add 1 mL of 0.5 mol / L acetic acid aqueous solution for every 1 g of pretreated jellyfish raw material, add 2.5% pepsin, sonicate at a frequency of 65 KHz for 75 min, extract at 4℃ for 3 h, centrifuge, take the supernatant to obtain crude collagen, dialyze in 0.02 mol / L Na2HPO4 solution for 24 h, freeze dry to obtain jellyfish collagen; (3) Dissolve the jellyfish collagen obtained in step (2) in water to form a jellyfish gel protein solution of 3.5 mg / mL; mix the jellyfish gel protein solution with CMP aqueous solution (1 μM / μL) at a volume ratio of 1:5, and then use 365 nm UV light (light intensity 10 mW / cm²) to test the mixture. 2 Activation, incubation at 4 ℃ for 3 h, yielding collagen hybrid peptides; (4) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid were homogenized in a mass ratio of 5:1:20 using a microfluidic homogenizer. The ultrasonic-assisted treatment conditions were 400W ultrasonic power, 10min ultrasonic time, 85MPa microfluidic pressure and 8 microfluidic cycles to prepare a multi-component protein complex, which was used as a natural bio-ink. (5) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing. The printhead used is a multi-nozzle array nanoscale nozzle with a nozzle size of 235 nm, an operating voltage of 1350 V applied to the nozzle, an operating distance of 1.5 mm, an ink flow rate of 3 μL / min, and a substrate receiving speed of 25 mm / s.

[0039] Example 3 A method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field current fluid jetting includes the following steps: (1) Pre-treated jellyfish raw material: soaked in deionized water 3 times, 2 hours each time, and pulped; add 15 mL of 0.1 mol / L sodium hydroxide aqueous solution per 1 g of jellyfish, soak for 3 days, wash with water until neutral, and obtain pre-treated jellyfish raw material; (2) Take the pretreated jellyfish raw material, add 2 mL of 0.5 mol / L acetic acid aqueous solution for every 1 g of pretreated jellyfish raw material, add 1.5% pepsin, sonicate at a frequency of 60 KHz for 80 min, extract at 4℃ for 5 h, centrifuge, take the supernatant to obtain crude collagen, dialyze in 0.02 mol / L Na2HPO4 solution for 24 h, freeze dry to obtain jellyfish collagen; (3) Dissolve the jellyfish collagen obtained in step (2) in water to form a 4 mg / mL jellyfish gel protein solution; mix the jellyfish gel protein solution with CMP aqueous solution (1.5 μM / μL) at a volume ratio of 1:10, and then use 365 nm UV light (light intensity 10 mW / cm²) to test the mixture. 2 Activation, incubation at 4 ℃ for 3 h, yielding collagen hybrid peptides; (4) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid were homogenized in a ratio of 8:1:35 using a microfluidic homogenizer. The ultrasonic-assisted treatment conditions were 400 W ultrasonic power, 18 min ultrasonic time, 125 MPa microfluidic pressure and 6 microfluidic cycles to prepare a multi-component protein complex, which was used as a natural bio-ink. (5) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing. The printhead used is a multi-nozzle array nanoscale nozzle with a nozzle size of 250 nm, an operating voltage of 1400 V applied to the nozzle, an operating distance of 1.6 mm, an ink flow rate of 5 μL / min, and a substrate receiving speed of 25 mm / s.

[0040] Example 4 A method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field current fluid jetting includes the following steps: (1) Pre-treated jellyfish raw material: soaked in deionized water 3 times, 2.5 h each time, and pulped; add 25 mL of 0.1 mol / L sodium hydroxide aqueous solution per 1 g of jellyfish, soak for 2 days, wash with water until neutral, and obtain pre-treated jellyfish raw material; (2) Take the pretreated jellyfish raw material, add 2 mL of 0.5 mol / L acetic acid aqueous solution for every 1 g of jellyfish raw material, add 1% pepsin, sonicate at a frequency of 65 KHz for 80 min, extract at 4 ℃ for 2.5 h, centrifuge, take the supernatant to obtain crude collagen, dialyze in 0.02 mol / L Na2HPO4 solution for 24 h, freeze dry to obtain jellyfish collagen; (3) Dissolve the jellyfish collagen obtained in step (2) in water to form a jellyfish gel protein solution of 3.9 mg / mL; mix the jellyfish gel protein solution with CMP aqueous solution (1.3 μM / μL) at a volume ratio of 1:7, and then use 365 nm UV light (light intensity 10 mW / cm²) to test the mixture. 2 Activated, and incubated at 4 ℃ for 3 h to obtain collagen hybrid peptides; (4) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid were homogenized in a mass ratio of 5:1:25 using a microfluidic homogenizer. The ultrasonic-assisted treatment conditions were 500 W ultrasonic power, 10 min ultrasonic time, 110 MPa microfluidic pressure and 7 microfluidic cycles to prepare a multi-component protein complex, which was used as a natural bio-ink. (5) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing. The printhead used is a multi-nozzle array nanoscale nozzle with a nozzle size of 230 nm, an operating voltage of 1450 V applied to the nozzle, an operating distance of 2 mm, an ink flow rate of 6 μL / min, and a substrate receiving speed of 35 mm / s.

[0041] Comparative Example 1 Same as Example 1, except that there is no ultrasound assistance in step (2).

[0042] Quantitative calculation of the extracted jellyfish collagen content revealed that the collagen extraction rate was lower than that in Example 1.

[0043] Low extraction rate and purity will limit the wound healing function of the dressings prepared subsequently. This is because there are fewer sites for pepsin reaction without microwave assistance, resulting in low extraction efficiency. Even with long-term acid-enzyme extraction, the extraction rate remains low.

[0044] Comparative Example 2 Same as Example 1, except that in step (2), the extraction is performed using trypsin or alkaline protease.

[0045] Molecular weight determination of the extracted jellyfish collagen revealed that the molecular weight of collagen extracted by pepsin was significantly larger than that extracted by trypsin or alkaline protease. Proteins with excessively small molecular weights cannot achieve a complete three-dimensional structure, leading to functional localization.

[0046] Comparative Example 3 Same as Example 1, except that there is no microjet homogenization in step (4).

[0047] The resulting collagen dressing has a slower wound healing speed and poorer mechanical strength. This may be because the jellyfish collagen, hyaluronic acid, and collagen hybrid peptides are thoroughly and evenly mixed through microfluidics to obtain a blend system with a small average particle size and low PDI. The composite material combines the advantages of natural and synthetic materials, providing higher mechanical strength and good specific binding ability while ensuring biocompatibility.

[0048] Comparative Example 4 Same as Example 1, except that the mass ratio of jellyfish collagen, collagen hybrid peptide and hyaluronic acid in step (4) is 20:15:15. The obtained collagen dressing had lower air permeability, tensile strength, and mechanical strength than the example group, possibly due to an unreasonable ratio that resulted in higher viscosity of the bio-ink, which could not guarantee the physical properties of the gel dressing.

[0049] Comparative Example 5 Same as Example 1, except that the printing conditions in step (5) are as follows: the ink syringe is loaded into the 3D printer; then the 3D printing of the collagen gel dressing with a mesh structure is carried out according to the printing parameters of the nozzle moving speed of 3.17 mm / s, the nozzle extrusion distance of 0.35 mm, and the nozzle moving speed of 4.83 mm / s, so as to obtain the jellyfish collagen dressing.

[0050] The resulting collagen dressing was thin and had poor mechanical strength, possibly because the mesh structure could only ensure the breathability of the gel dressing, and its high degree of integration resulted in weak resistance to local damage.

[0051] Test case (1) Extraction rate and purity of jellyfish collagen: The hydroxyproline content in the extracted jellyfish collagen was determined using a hydroxyproline kit. The extraction rate was calculated using the following formula: T = A / B × 100% A and B are the hydroxyproline content in the extract and the hydroxyproline content in the jellyfish raw material, respectively. Figure 1 The extraction rate of jellyfish collagen obtained in Comparative Example 1 and Example 1 was calculated by... Figure 1 It is evident that pepsin has the highest collagen extraction rate, reaching 33.69±1.23%.

[0052] Purity was determined by the Kjeldahl method: Weigh 0.20 g of solid sample and transfer it to a dry 100 mL nitrogen determination flask. Add 0.2 g of copper sulfate, 6 g of potassium sulfate, and 20 mL of sulfuric acid. Shake slightly and place a small funnel at the mouth of the flask. Support the flask at a 45° angle against a perforated asbestos mesh. Heat carefully until the contents are completely carbonized and the foaming stops completely. Increase the heat and maintain a gentle boil until the liquid turns a clear, transparent blue-green color. Continue heating for another 0.5–1 h. Remove from heat and allow to cool. Carefully add 20 mL of water. After cooling, transfer the solution to a 100 mL volumetric flask and rinse the nitrogen determination flask with a small amount of water. Add the rinsing solution to the volumetric flask and add water to the mark. Mix well and set aside. Perform a reagent blank test simultaneously. Assemble the nitrogen determination apparatus. Fill the steam generator flask with water to 2 / 3 full, add several glass beads, a few drops of methyl red indicator solution, and a few milliliters of sulfuric acid to maintain acidity. Use a pressure regulator to control the temperature and heat the water in the steam generator flask to boiling. Add 10 mL of boric acid solution (20 g / L) and 1-2 drops of mixed indicator solution to the receiving flask, ensuring the lower end of the condenser is submerged below the liquid surface. Accurately pipette 10 mL of the sample treatment solution into the reaction chamber through a small funnel. Rinse the small beaker with 10 mL of water to allow the solution to flow into the reaction chamber. Seal the small beaker tightly with a rod-shaped glass stopper. Pour 10 mL of sodium hydroxide solution (400 g / L) into the small beaker, lift the stopper to allow it to slowly flow into the reaction chamber, and immediately seal the beaker tightly. Add water to the small beaker to prevent leakage. Clamp the screw clamp and begin distillation. Distill for 5 minutes. Move the receiving flask until the liquid level is above the lower end of the condenser, and distill for another 1 minute. Then rinse the outside of the lower end of the condenser with a small amount of water. Remove the receiving flask. Titrate with a standard sulfuric acid or hydrochloric acid solution (0.05 mol / L) until the endpoint is gray or blue-purple. Simultaneously, accurately pipette 10 mL of reagent blank digestion solution and repeat the above procedure.

[0053] The formula for calculating the protein content in a sample is as follows:

[0054] In the formula: X—The protein content in the sample, expressed in grams per 100 grams (g / 100g); V1—The volume of hydrochloric acid standard titrant consumed by the sample, in milliliters (mL); V2—The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the reagent blank, in milliliters (mL); C—Concentration of hydrochloric acid standard titration solution, in moles per liter (mol / L); The mass of nitrogen equivalent to 0.0140–1.0 mL of hydrochloric acid [c(HCl) = 1.000 mol / L] standard titration solution, in grams (g). m—mass of the sample, in grams (g); F—the coefficient for converting nitrogen to protein. It is typically 6.25. The purity of the extracted jellyfish collagen was measured to be 79.94 ± 2.44%.

[0055] (2) Jellyfish collagen SDS-PAGE gel electrophoresis: Add 20 μL of the prepared collagen sample to an SDS-PAGE precast gel made of 8% separating gel and 5% stacking gel, and electrophoresis at 120 V for 60 min. After electrophoresis, stain the precast gel with Coomassie Brilliant Blue R-250 rapid staining solution for 30 min, and then destain.

[0056] Depend on Figure 2 It can be seen that the molecular weight of jellyfish collagen extracted by pepsin is significantly larger than that of collagen extracted by other enzymes. (3) Physicochemical properties of collagen gel plaster: The method for determining tensile strength is as follows: The dry and wet test materials are cut into standard strips according to GB / T634-1996, and the tensile strength of the specimens is determined on the Instron universal testing machine in the United States.

[0057] The method for determining air permeability is as follows: A certain amount of distilled water is placed in a wide-mouth bottle, and the bottle mouth is sealed with the material to be tested. Another unsealed wide-mouth bottle containing distilled water is used as a control group. The bottle is left at room temperature for 24 hours, and the air permeability is calculated using the following formula: Air permeability = 24h water loss of experimental group / water loss of control group × 100%.

[0058] The method for determining water absorption rate is as follows: Cut the material to be tested into 1cm×1cm samples, accurately weigh the mass W1, immerse it in a container containing 50mL of deionized water or physiological saline, and let it stand at room temperature until it is saturated with water; after taking it out, use filter paper to absorb the surface moisture, accurately weigh the mass W2, and calculate the water absorption rate. The calculation formula is: Water absorption rate = (W2 - W1) / W1 × 100%.

[0059] The method for determining the moisture retention rate is as follows: Cut the material to be tested into 2cm×2cm samples, accurately weigh the mass W1, immerse it in deionized water until it absorbs water and reaches equilibrium, remove it, centrifuge at 500r / min for 3min, accurately weigh the mass W2, and calculate the moisture retention rate. The calculation formula is: Moisturizing rate = (W1 - W2) / W1 × 100%.

[0060] The test results are shown in Table 1. The table shows that the printed dressing from the embodiment exhibits superior physical properties. Firstly, the jellyfish collagen extracted from the formula retains more complete bioactivity and overcomes heat sensitivity defects; the three-component composite system results in higher mechanical strength. Secondly, thanks to the larger swelling space and contact area of ​​the 3D scaffold structure, the printed dressing can absorb moisture or wound exudate more quickly than ordinary dressings.

[0061] Table 1

[0062] (4) In vitro cytotoxicity assay (CCK-8 assay): The study included a control group (L929 mouse fibroblasts) and an experimental group (3D-printed medical dressings and L929 mouse fibroblasts). Before cell implantation, the dressing was soaked in 70% ethanol solution for 2 hours, then washed five times with PBS, and each side of the dressing was irradiated under UV light for 30 minutes. The materials were then immersed in culture medium overnight at 37°C. Subsequently, 500 μL of L929 cell suspension was added to each well, and the wells were incubated at 37°C, 5% CO2, and 95% humidity.

[0063] The proliferation of L929 cells on a dressing was evaluated using the CCK-8 cell proliferation assay kit. At predetermined time points, the CCK-8 stock solution was diluted 1:10 (v / v) with culture medium to obtain the working solution. The culture medium was removed from the wells, and 200 μL of the working solution was added to each well. After incubation in the dark for 2 hours, the cells were gently shaken to mix. Then, 100 μL of the supernatant working solution was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader. The OD values ​​were recorded. Depend on Figure 4 The OD values ​​of the example group were significantly higher than those of the control group, proving that the material has no cytotoxicity and good biocompatibility.

[0064] (5) Wound healing rate experiment: The experiment used 8-10 week old female SD rats purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China), and all procedures were performed according to relevant regulations. SD rats were anesthetized by intraperitoneal injection of 10% hydroxyl chloride hydrate at a dose of 0.3 ml per 100 g body weight, followed by a 2 cm square full-thickness incision on each side. The dressing was made by removing the skin from the back of each rat. The collagen dressing of this invention was then applied to each wound. Uncovered wounds served as a blank control group. The material was fixed with sterile gauze and then bandaged with tape. The rats were then individually caged. The dressing was changed on postoperative days 3, 7, 10, and 14.

[0065] On postoperative days 3, 7, 10, 14, and 17, the wound was photographed, the wound area was calculated, and the wound closure rate was quantified using the following formula: Wound closure rate = (W0 - W) t ) / W0×100%.

[0066] Among them, W0 and W t The wound areas are on postoperative day 0 and day n (n = 3, 7, 10, 14 and 17), respectively.

[0067] Depend on Figure 5 It was found that the wounds treated with the dressing in the example group healed significantly faster than those in the control group. The wounds were clearly closed after 10 days, and almost completely healed by the 14th day. The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a jellyfish collagen hydrogel dressing based on electrostatic field electrofluid jetting, characterized in that, Includes the following steps: (1) After pretreatment of jellyfish raw materials, collagen was extracted by ultrasound-assisted acid-enzyme complex extraction to obtain jellyfish collagen; (2) Collagen hybrid peptides were prepared by activating cage-like collagen mimic peptide CMP with ultraviolet light and binding it to jellyfish collagen. (3) The jellyfish collagen obtained in step (1), the collagen hybrid peptide obtained in step (2) and hyaluronic acid are uniformly mixed by a high-pressure microfluidic homogenizer to prepare a multi-component protein complex system as a natural bio-ink; the mass ratio of jellyfish collagen, collagen hybrid peptide and hyaluronic acid is 5-15:1-2:20-40. (4) Under sterile vacuum conditions, electrostatic field current fluid jet 3D printing technology is used to print natural biological ink layer by layer into a three-dimensional porous structure model with an irregular truss structure using high-resolution micro-nano nozzles. Collagen hybrid peptides are embedded in the truss structure to obtain the jellyfish collagen hydrogel dressing.

2. The preparation method according to claim 1, characterized in that, In step (1), the method for pretreatment of jellyfish raw material is as follows: jellyfish raw material is mixed with sodium hydroxide aqueous solution, stirred for 1-3 days, centrifuged, and the precipitate is washed with deionized water until neutral to obtain pretreated jellyfish raw material.

3. The preparation method according to claim 1, characterized in that, In step (1), the method of ultrasound-assisted acid-enzyme complex extraction of collagen is as follows: pretreated jellyfish raw material is mixed with acetic acid aqueous solution, pepsin is added, and then ultrasonic extraction is performed for 2-6 hours; freeze drying is performed to obtain jellyfish collagen. The concentration of the acetic acid aqueous solution is 0.5 mol / L; The frequency of the ultrasound is 60-70 kHz, and the duration of the ultrasound is 60-100 min. The mass-to-volume ratio of the pretreated jellyfish raw material to the acetic acid aqueous solution was 1:1-2 g / mL; The amount of pepsin used is 1-3% of the mass of the pretreated jellyfish raw material.

4. The preparation method according to claim 1, characterized in that, In step (2), the jellyfish collagen obtained in step (1) is dissolved in water to form a jellyfish collagen solution of 3.5-4 mg / mL; the jellyfish collagen solution is mixed with CMP aqueous solution, then activated by ultraviolet light and incubated at 4 ℃ for 2-4 h.

5. The preparation method according to claim 4, characterized in that, In step (2), the volume ratio of CMP aqueous solution to jellyfish collagen solution is 1:5-10.

6. The preparation method according to claim 1, characterized in that, In step (3), the parameters of the high-pressure microjets homogenizer are as follows: the ultrasonic-assisted processing conditions are ultrasonic power of 400-500 W, ultrasonic time of 10-20 min, microjets pressure of 75-125 MPa, and microjets 6-9 times.

7. The preparation method according to claim 1, characterized in that, In step (4), the working voltage of electrostatic fluid jetting is 1300-1500 V, the working distance is 1.5-2 mm, the ink flow rate is 1-7 μL / min, and the receiving substrate speed is 20-40 mm / s.

8. The preparation method according to claim 1, characterized in that, In step (4), the nozzle is a multi-nozzle array nanoscale nozzle with a nozzle size of 200-250 nm.

9. A hydrogel dressing for jellyfish collagen based on electrostatic field electrofluid jetting, prepared by the method according to any one of claims 1-8.

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