Multifunctional wound dressing with yin-yang membrane structure and preparation method thereof

Abstract

The application discloses a yin-yang structure wound dressing, which comprises two layers, wherein the upper layer is a dense hydrophobic layer, which is a polytrimethylene carbonate fiber membrane and simulates an epidermis structure; and the lower layer is a hydrophilic layer, which is a natural high molecular fiber hydrogel membrane and simulates a dermis structure. Meanwhile, the application also discloses a preparation method of the yin-yang structure wound dressing. The multifunctional wound dressing with the yin-yang membrane structure is prepared by firstly adopting electrospinning technology to prepare a natural high molecular fiber hydrogel membrane and a polytrimethylene carbonate fiber membrane with two different properties, and then bonding the two membranes with different properties together by a photo-crosslinking method to construct a multi-level wound repair dressing with a stable structure and simulating skin, so that the nanofiber structure of human skin is restored to the maximum extent, and the purpose of promoting the growth of granulation tissue at a wound and the recovery of structures such as hair follicles, blood vessels and nerves is achieved from the structure of the skin itself.

Description

Technical Field

[0001] This invention belongs to the field of biomedical devices, specifically relating to a multifunctional wound dressing with an anisotropic membrane structure and its preparation method. Background Technology

[0002] The skin performs vital physiological functions but is also highly susceptible to injury. For full-thickness skin defects caused by injury, relying solely on the skin's own repair capabilities takes a very long time to heal. Achieving rapid healing and restoring the physiological functions of full-thickness skin defects is a significant clinical challenge. Skin wounds are complex issues involving numerous physiological responses. Studies have shown that maintaining local wound moisture can better promote granulation tissue growth and skin tissue remodeling. During wound healing, exogenous bacterial infections are prone to occur, leading to inflammation and delaying or even disrupting the healing process.

[0003] Currently, the main method to promote wound healing is the use of various wound dressings, such as adhesive bandages. However, traditional wound dressings like adhesive bandages and bandages often only provide simple physical isolation and do not effectively regulate the wound microenvironment, thus failing to significantly promote wound healing. Hydrogel dressings are also being extensively studied, but they tend to dehydrate and dry quickly, delaying wound healing. Therefore, the development of novel, multifunctional wound dressings has attracted widespread attention.

[0004] Currently, polymeric materials used in biomedicine can be divided into two categories based on their source: natural polymers and synthetic polymers. Each type has its own advantages and disadvantages in use. Natural polymers can often be obtained directly from nature. By processing various collagens, celluloses, and polysaccharides found in nature, their structural units are obtained, and after purification, we get gelatin, various types of cellulose, chitosan, hyaluronic acid, and other materials commonly used in the biomedical field. These naturally obtained macromolecular materials have superior biocompatibility due to their simple acquisition methods, and can be applied in the field of biomaterials with simple processing. For example, gelatin is a natural material that has been widely used in wound repair. As partially hydrolyzed collagen residues, gelatin is homologous to protein fibers in the skin. Its rich protein reserves and numerous RGB sequences on its macromolecular chains have a good promoting effect on wound repair. At the same time, as a hydrophilic natural polymer, its cross-linked products have hydrogel properties, which can maintain wound hydration for a short time when used as a dressing.

[0005] The disadvantages of the above-mentioned prior art include: (1) Commercial dressings can only play a simple physical isolation role and have a limited effect on promoting repair, but when they are removed, they will cause secondary damage to the wound, further increasing the difficulty of wound repair. (2) Common hydrogel dressings tend to lose moisture easily and need to be applied to the wound multiple times to promote wound repair. In this process, in order to ensure the repair effect, it may be necessary to open the wound, which can easily cause secondary damage. Summary of the Invention

[0006] The purpose of this invention is to provide a multifunctional wound dressing with a yin-yang membrane structure and its preparation method.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A wound dressing with a "yin-yang" structure. This dressing comprises two layers: an upper, dense hydrophobic layer, a polytrimethylene carbonate (PTMC) fiber membrane with a biomimetic epidermal structure, which isolates and filters exogenous bacterial infections and provides mechanical support; and a lower, hydrophilic layer, a natural polymer fiber hydrogel membrane, mimicking the dermal layer structure, designed to adhere to the wound site, maintain a moist microenvironment, and promote wound healing. The PTMC fiber membrane of the hydrophobic layer and the natural polymer fiber hydrogel membrane of the hydrophilic layer are cross-linked through chemical bonds at their interface, forming a stable structure.

[0009] The material of natural polymer fiber hydrogel membranes is a photocrosslinkable natural polymer, specifically, it can be at least one of gelatin, collagen, and silk fibroin, or a composite material formed by mixing at least two of these three materials, or a mixture of these materials with other natural polymer materials to form gelatin-based composite materials, collagen-based composite materials, or silk fibroin-based composite materials. Other natural polymer materials include chitosan, hyaluronic acid, and sodium alginate.

[0010] The hydrophobic layer consists of densely packed PTMC fibers with a thickness of 10–400 μm, fiber diameters ranging from 0.5 to 2 μm, and pore sizes ≤1 μm. The hydrophilic layer is a fiber membrane with a thickness of 20–500 μm, fiber diameters mainly ranging from 0.1 to 2 μm, and pore sizes ranging from 10 to 150 μm.

[0011] Natural polymer fiber hydrogel membranes can also contain exosomes, active small molecules, traditional Chinese medicine ingredients, active peptides, etc., to promote wound repair.

[0012] A method for preparing a multifunctional wound dressing with a yin-yang membrane structure includes the following steps:

[0013] (1) Preparation of natural polymer fiber hydrogel membrane with hydrophilic layer

[0014] A photocrosslinkable natural polymer material is formulated into a spinning solution, a photoinitiator is added to the spinning solution, and a natural polymer fiber hydrogel membrane is prepared by electrospinning technology.

[0015] (2) Electrospinning PTMC fibers onto natural polymer fibers

[0016] Photocrosslinkable PTMC and high molecular weight PTMC are mixed in an appropriate ratio and prepared into a spinning solution. A photoinitiator is added to the spinning solution, and the spinning solution is sprayed onto the natural polymer fiber hydrogel membrane prepared in step (1) by electrospinning technology to obtain a bilayer membrane with a hydrophilic layer and a hydrophobic layer.

[0017] (3) Photocrosslinked bilayer film

[0018] Prepare an ethanol solution containing a photoinitiator as a crosslinking solution. Completely immerse the obtained bilayer membrane in the crosslinking solution. After crosslinking is completed, remove the bilayer membrane from the crosslinking solution, rinse the membrane material with ethanol to remove residual photoinitiator, then rinse with deionized water to remove ethanol and freeze-dry to obtain a structurally stable anion-ion bilayer dressing.

[0019] In step (1):

[0020] The photoinitiator has a mass fraction of 0.1% to 5% relative to the natural polymer material.

[0021] The thickness of the natural polymer fiber hydrogel membrane is controlled between 20 and 500 μm.

[0022] Synthesis of photocrosslinkable natural polymer materials: The materials are obtained by reacting methacrylic anhydride with natural polymer materials, with the grafting degree controlled within the range of 30% to 95%.

[0023] Natural polymer materials include gelatin, collagen, or silk fibroin.

[0024] The chemical reaction equations for the above process are as follows:

[0025]

[0026] In step (2):

[0027] The mass ratio of photocrosslinkable PTMC to high molecular weight PTMC is 1% to 10%.

[0028] The photoinitiator has a mass fraction of 0.1% to 5% relative to the spinning solution.

[0029] The thickness of PTMC fiber membranes ranges from 10 to 400 μm.

[0030] Synthesis of photocrosslinkable PTMC:

[0031] Trimethylolpropane and stannous octoate were used as initiator and catalyst, respectively, to initiate the ring-opening polymerization of trimethylene carbonate (TMC) monomers at 140°C, yielding three-arm polytrimethylene carbonate (PTMC) oligomers. Subsequently, methacrylic anhydride and the PTMC oligomers underwent a chemical reaction to ultimately obtain photocrosslinkable PTMC. The molecular weight of the synthesized photocrosslinkable PTMC was controlled between 10 and 50 kDa, with a grafting rate of 50% to 80%.

[0032] The chemical reaction formulas for the above process are as follows:

[0033]

[0034] Synthesis of high molecular weight PTMC:

[0035] High molecular weight PTMC was synthesized via bulk polymerization of trimethylene carbonate. The synthesized high molecular weight PTMC had a molecular weight of 100–500 kDa. The chemical reactions involved in the process are as follows:

[0036]

[0037] The photoinitiator of the present invention is selected from 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (IR2959), lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP), etc., with IR2959 being preferred.

[0038] The present invention has the following advantages and effects compared with the prior art:

[0039] 1. The multifunctional wound dressing with a yin-yang membrane structure of the present invention uses two materials with different properties, natural polymer and PTMC, and combines them with electrospinning to construct a biomimetic multi-layered wound repair dressing. The electrospinning method can maximize the restoration of the original skin fiber structure, so as to promote the growth of granulation tissue and the recovery of structures such as hair follicles, blood vessels and nerves at the wound site from the perspective of the skin's own structure. At the same time, it is expected that exosomes, growth factors or small molecules of traditional Chinese medicine can be added as cell-active substances, which can promote cell proliferation and migration at the wound site through paracrine action, while inhibiting the inflammatory response, thereby promoting wound repair.

[0040] 2. This invention utilizes electrospinning technology, resulting in a fiber pad with a microporous structure. This structure maintains the necessary coverage and protection for the wound while also providing breathability. Furthermore, the nonwoven fabric obtained through electrospinning can, to some extent, mimic the structure of human skin; the interwoven fibrous structure closely resembles the dense collagen fiber arrangement of human skin. This method also allows for the treatment of the fiber pad's surface microstructure, endowing it with superior properties such as superhydrophobicity and antibacterial properties.

[0041] 3. The polytrimethylene carbonate (PTMC) used in this invention has a glass transition temperature of -20℃ and is a type of polymer material with excellent elasticity. Furthermore, PTMC has a unique degradation mechanism; it is essentially non-hydrolyzed in vitro, but after implantation, it rapidly degrades through surface erosion by lipases, producing CO2 and H2O as degradation products, which do not trigger inflammatory responses.

[0042] 4. The outer layer of the dressing of this invention uses a PTMC fiber membrane. Compared with materials used in existing technologies (such as polyvinylidene fluoride (PVDF), polycaprolactone, polylactic acid, PLGA, etc.), PTMC material itself has the following unique advantages: First, an important characteristic of PTMC material is its biodegradability. Mediated by lipase enzymes present in the wound, the material is completely absorbed after skin repair. Second, PTMC material does not produce acidic byproducts after degradation and will not cause wound inflammation. Third, the PTMC fiber membrane of the outer layer of the dressing has a dense microstructure (small pore size and high porosity), which, while ensuring breathability, can also filter bacteria to a certain extent, playing a passive antibacterial role, thereby ensuring that the wound is not infected by bacteria during the repair process. Compared with traditional antibacterial methods (which often involve adding additional antibacterial substances (such as silver, antibiotics, antimicrobial peptides, etc.), this technology does not require the addition of additional drugs, is more environmentally friendly, simpler to prepare, and lower in cost.

[0043] 5. The inner layer of the dressing of this invention is a natural polymer fiber hydrogel material such as gelatin. Compared with the polyethylene (PVA) layer used in previously reported yin-yang dressings, its composition is closer to that of the natural extracellular matrix, which can provide a better microenvironment for cell migration and growth, thereby better promoting skin wound repair. Exosomes, active small molecules, and traditional Chinese medicine ingredients can be added to the inner layer of the dressing to promote wound healing.

[0044] 6. The outer PTMC fiber membrane of the dressing of this invention is a hydrophobic layer, while the inner layer is a hydrophilic layer made of natural polymer fiber hydrogels such as gelatin. The interface between the inner and outer layers is bonded by chemical bonds to form a stable structure. This combination of two membranes can better lock in moisture, reduce water loss, maintain local wound hydration, and promote wound healing. The difference compared with existing yin-yang membrane dressing technology is that the hydrophobic layer in this invention, by adjusting the microscopic topology of the fibers, achieves a contact angle of 81.3°±3° for the PTMC hydrophobic layer, making its hydrophilicity closer to that of natural skin (the contact angle of the surface layer of human skin is 80-100°).

[0045] 7. The dressing of this invention can create a better microenvironment for skin wound repair, with the following specific advantages:

[0046] It has excellent moisturizing properties, keeping wounds hydrated and promoting skin repair. It uses PTMC fibers as a hydrophobic layer and natural polymer fiber hydrogels such as gelatin as a hydrophilic layer; this bilayer interface forms a stable structure through chemical bond cross-linking.

[0047] Antibacterial action is achieved through physical filtration and isolation. By adjusting the pore size of PTMC fibers, bacteria can be filtered and isolated during wound healing without the need for additional medications, making it more environmentally friendly, simpler to prepare, and lower in cost.

[0048] The enzymatic degradation properties of the double-layer dressing allow it to be completely absorbed by the body after wound healing. The degradation products are non-acidic and will not cause wound inflammation, thus promoting wound healing. The outer layer is made of PTMC fiber, which can be degraded by lipases in the wound, while the inner layer, made of gelatin and other natural polymer fiber hydrogels, can be degraded by endogenous collagenases in the human body. The degradation products have no toxic side effects. Attached Figure Description

[0049] Figure 1 It is photocrosslinkable gelatin 1 The double peaks within the red circle in the 1H-NMR nuclear magnetic resonance spectrum indicate that the reaction was successful.

[0050] Figure 2 It is a photocrosslinkable PTMC 1 The double peaks (h and i) in the 1H-NMR spectrum indicate successful grafting.

[0051] Figure 3 This is a comparison of the microstructures of the anion-and-yin-yang membrane structure dressings. Janus is the anion-and-yin-yang membrane structure dressing of this invention. (a) is a microstructure diagram of the hydrophilic layer Gelatin fiber; (b) is a microstructure diagram of the hydrophobic layer PTMC fiber membrane; (c) is a cross-sectional microstructure diagram of the anion-and-yin-yang membrane structure dressing.

[0052] Figure 4Statistical analysis was performed on the fiber diameter and pore size of the hydrophilic Gelatin fiber membrane and the hydrophobic PTMC fiber membrane, respectively. (a) Statistical analysis of the fiber diameter of the hydrophilic Gelatin fiber membrane; (b) Statistical analysis of the pore size of the hydrophilic Gelatin fiber membrane; (c) Statistical analysis of the fiber diameter of the hydrophobic PTMC fiber membrane; (d) Statistical analysis of the pore size of the hydrophobic PTMC fiber membrane.

[0053] Figure 5 This is a comparison of the water retention performance of the yin-yang membrane structure dressing of the present invention. Janus is the yin-yang membrane structure dressing of the present invention. (a) is an overview of the 21-day water loss experiment; (b) is the daily water loss statistics; (c) is the 21-day water loss curve.

[0054] Figure 6 This is a comparison of the tensile properties of different membrane materials under different conditions. Janus is the yin-yang membrane structure dressing of this invention. (a) is the tensile curve under wetting conditions; (b) is the elastic modulus under wetting conditions; (c) is the elongation at break under wetting conditions.

[0055] Figure 7 Contact angle tests of the hydrophobic and hydrophilic layers of the anion-and-hypothesis membrane structure dressing, Janus being the anion-and-hypothesis membrane structure dressing of this invention. (a) Contact angle image; (b) Quantitative statistics of contact angle size.

[0056] Figure 8 Different membrane materials exhibit varying permeability to bacteria. Janus is the yin-yang membrane structure dressing of this invention. (a) Schematic diagram of the experimental apparatus for testing bacterial barrier properties; (b) Graph showing the number of bacterial colonies on the plate of bacterial suspension collected from different bacterial collection bottles; (c) Quantitative statistics of residual Escherichia coli colonies; (d) Quantitative statistics of residual Staphylococcus aureus colonies; (e) Janus membrane cytotoxicity test.

[0057] Figure 9 Representative images of wound area from different groups on days 0, 4, 7, 10, and 15.

[0058] Figure 10 Animal experiments showed that Janus is the yin-yang membrane structure dressing of this invention. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0060] Example

[0061] A multifunctional wound dressing with a biomimetic yin-yang membrane structure, used for wound repair, is manufactured using the following steps:

[0062] (1) Synthesis of photocrosslinkable gelatin

[0063] Preheat deionized water to approximately 40°C. Add 10% gelatin to the aqueous solution and maintain the temperature at 40°C while stirring thoroughly to dissolve. When the system becomes clear and pale yellow, add methacrylic anhydride (Sigma-Aldrich, catalog number 276685-100mL) dropwise at a volume-to-gelatin mass ratio of 1.6mL / g, controlling the addition rate at 0.5mL / min. After complete addition, the system will be white and turbid. Continue heating to 50–60°C and react for 4–6 hours. After the reaction is complete, turn off the heating and allow it to cool to room temperature. Dilute the reaction solution with an equal volume of 5×PBS buffer to terminate the reaction. Pre-treat a dialysis bag (molecular weight cutoff 7000kDa) by boiling. Add the reacted solution to the dialysis bag and dialyze at 40°C in deionized water for 7 days, changing the dialysate twice daily. After dialysis, remove the solution and freeze-dry it to obtain the sample. Dissolve a small amount of the solid in deuterated water and use 1H NMR spectroscopy to confirm successful grafting. Figure 1 The grafting rate was calculated to be around 90% based on the peak intensity of the spectrum.

[0064] (2) Synthesis of photocrosslinkable PTMC and high molecular weight PTMC

[0065] Photocrosslinkable PTMC synthesis: 10g of trimethylene carbonate (TMC, Huasubao, catalog number 9990100000) was heated at 80℃ and stirred until melted, presenting as a watery transparent liquid. Under nitrogen protection, 10.4μl of stannous isooctanoate (Sn(Oct)2, Sigma-aldrich, catalog number S3252) and 0.09g of trimethylolpropane (Sigma-aldrich, catalog number 93370) were added dropwise. Under nitrogen protection, the temperature was raised to 140℃ and the polymerization reaction was carried out for one day. The polymerized PTMC was a transparent, soft solid. Heating was stopped, and 30–40 mL of dry dichloromethane was added to dissolve the polymer. After complete dissolution, a colorless, clear solution was obtained. Under nitrogen protection, 2 mL of triethylamine (TEA, Sigma-Aldrich, catalog number 471283-500 mL), 2.4 mL of methacrylic anhydride (Sigma-Aldrich, catalog number 276685-100 mL), and 10.6 mg of hydroquinone (Sigma-Aldrich, catalog number H9003-100 G) were added. The reaction was continued at room temperature, protected from light, and under nitrogen protection. After four days of reaction, the reactants were removed, and unreacted water-soluble impurities were removed by repeated extraction with deionized water. The precipitate was then precipitated in cold methanol, washed with cold methanol and cold ethanol to remove other impurities, and dried in a vacuum oven to obtain a white solid product. A small amount of the solid was dissolved in deuterated chloroform, and proton NMR spectroscopy confirmed successful grafting. Figure 2Based on the results, the molecular weight was calculated to be approximately 15100 g / mol, with a grafting rate of 80%.

[0066] Synthesis of high molecular weight PTMC: A small amount of TMC monomer was placed in a reaction flask, and linear high molecular weight PTMC was carried out by ring-opening polymerization at 140℃ using stannous isooctanoate as a catalyst. The reaction flask was sealed under vacuum for 3 days. The product was dissolved in sufficient chloroform and precipitated in cold ethanol. The product was washed with ethanol, and excess ethanol was removed by vacuum drying oven. The final high molecular weight PTMC obtained had a molecular weight of approximately 500 kDa.

[0067] (3) Preparation of gelatin spinning solution

[0068] Photocrosslinkable gelatin was dissolved in hexafluoroisopropanol (HFIP) for 24 hours to obtain a spinning solution with a concentration of 20%. Then, IR2959 (sigma-aldrich, catalog number 410896-50G) at a mass fraction relative to gelatin was added as a photoinitiator, and stirring was continued for 30 minutes to obtain the gelatin spinning solution.

[0069] (4) Preparation of PTMC spinning solution

[0070] Photocrosslinkable PTMC (molecular weight approximately 15,000) and high molecular weight PTMC (molecular weight approximately 500,000) were added to HFIP (mass ratio of high molecular weight PTMC to photocrosslinkable PTMC was 5%), stirred for 24 h to dissolve, and finally obtained a spinning solution with a polymer mass concentration of 5%. Then, IR2959 with a relative total polymer mass fraction of 1.5% was added as a photoinitiator, and stirring was continued for 30 min to obtain PTMC spinning solution.

[0071] (5) Spinning Janus double-layer dressing and ultraviolet crosslinking

[0072] First, gelatin was spun at a flow rate of 1.5 ml / h, a spinning voltage of 13 kV, and a spinning needle distance of 10 cm from the receiving plate for 40 minutes to obtain a gelatin fiber membrane. Then, PTMC was directly spun on top of this membrane at a flow rate of 1 ml / h, a spinning voltage of 18 kV, and a spinning needle distance of 10 cm from the receiving plate for 120 minutes to obtain a bilayer membrane material. During the spinning process, UV irradiation was maintained for pre-crosslinking to prevent the PTMC material from collapsing during casting.

[0073] Prepare an ethanol solution of IR2959 with a concentration of 0.5% wt. Immerse the obtained bilayer membrane completely in the crosslinking solution. Perform UV crosslinking on the Gelatin layer and the PTMC layer sequentially, with each layer crosslinking for 5-10 minutes. After crosslinking, remove the material from the spinning solution, rinse the membrane material with ethanol to remove residual IR2959, then rinse with deionized water to remove ethanol and freeze-dry to obtain a multifunctional wound dressing with an anion-ion membrane structure.

[0074] The following examples illustrate the preparation method of the gelatin-only membrane:

[0075] The spinning solution prepared according to step (3) of the embodiment was used for gelatin spinning. The spinning flow rate was 1.5 ml / h, the spinning voltage was 13 KV, the distance between the spinning needle and the receiving plate was 10 cm, and the spinning was carried out for 40 min to obtain a gelatin fiber membrane. An ethanol solution with a concentration of 0.5% wt of IR2959 was prepared as a crosslinking solution. The obtained gelatin fiber membrane was completely immersed in the crosslinking solution and crosslinked under ultraviolet light for 10 min. After the crosslinking was completed, the material was taken out from the crosslinking solution, and the membrane material was rinsed with ethanol to remove the residual IR2959. Then it was rinsed with deionized water to remove the ethanol and freeze-dried to obtain a single-layer gelatin fiber dressing (Gelatin Only membrane).

[0076] The following examples illustrate the preparation method of the PTMC-only membrane used:

[0077] The spinning solution prepared according to step (4) of the embodiment was used for spinning. The spinning flow rate was 1 ml / h, the spinning voltage was 18 kV, the distance between the spinning needle and the receiving plate was 10 cm, and the spinning was carried out for 120 min to obtain the membrane material. An ethanol solution of IR2959 with a concentration of 0.5% wt was prepared as the crosslinking solution. The obtained membrane material was completely immersed in the crosslinking solution and crosslinked under ultraviolet light for 10 min. After the crosslinking was completed, the material was taken out from the crosslinking solution, rinsed with ethanol to remove residual IR2959, then rinsed with deionized water to remove ethanol and freeze-dried to obtain a single-layer PTMC dressing (PTMC Only membrane).

[0078] Example 1

[0079] The differences in microstructure between the biomimetic double-layer dressings were compared, and the results are as follows: Figure 3 As shown.

[0080] Statistical results regarding fiber diameter and porosity of the Gelatin and PTMC layers of the Janus double-layer dressing are as follows: Figure 4 As shown.

[0081] Scanning electron microscopy revealed significant differences in the microstructure of the hydrophilic Gelatin layer and the hydrophobic PTMC layer of the electrospun Janus fiber membrane. The Gelatin layer exhibited a relatively loose fiber structure, primarily concentrated around 2 μm in diameter, with a wide porosity distribution ranging from 10 μm to 50 μm, closely resembling the protein fibers in the human dermis. This macroporous fiber membrane served as a support for the regeneration of local cells and granulation tissue at the wound site, promoting wound repair. The PTMC layer, on the other hand, had a relatively concentrated fiber diameter distribution, ranging from 0.9 to 1.8 μm. Furthermore, the connections between these fibers were very tight, similar to the tightly packed keratinocytes in the human epidermis, with numerous tiny pores visible on them. These small pores protected the wound, reducing the impact of the external environment on new tissue while maintaining the necessary permeability for wound repair. The resulting pore structure was significantly denser than that of the Gelatin layer, with pores distributed only between 0 and 1.65 μm, primarily concentrated in the 0.45–0.9 μm range.

[0082] Example 2

[0083] Comparing the water contact angles between the anion and ion membranes, and between the anion and ion membranes and the unbonded monolayer membrane, the results are as follows: Figure 7 As shown.

[0084] The single-layer PTMC (PTMC Only) membrane has poor hydrophilicity, and water molecules do not easily enter the gaps in the material. This is macroscopically manifested as a larger water contact angle (66.8°±3°). In contrast, the unbonded single-layer Gelatin (Gelatin Only) membrane has better hydrophilicity, and water molecules can more easily enter the gaps in the fiber membrane, resulting in a relatively smaller water contact angle (56.9°+3°).

[0085] When two membranes with different properties are combined, they influence each other. Specifically, their wettability deviates from each other. The PTMC material, which already has a large contact angle, further increases its contact angle to 81.3°±3°, which is similar to the contact angle of human skin under non-dry conditions. Meanwhile, the contact angle of gelatin further decreases to 48.4°±6°, exhibiting better wettability, thus achieving the purpose of biomimicry.

[0086] Example 3

[0087] To compare the water retention differences between bilayer and single-layer membranes, unbonded single-layer Gelatin-only membranes and Janus membranes were respectively used to cover the mouths of sample bottles containing equal volumes of deionized water. The bottles were then placed in a 37°C incubator, and the water loss rate in the sample bottles covered with different materials was recorded daily. The results are as follows: Figure 5 As shown.

[0088] Because the outer layer of a bilayer dressing with a Janus membrane structure is more hydrophobic and has lower porosity, it effectively reduces moisture loss from the underlying material and the wound during use, keeping the wound in a relatively moist state. Existing literature indicates that keeping the wound moist promotes wound healing and reduces scarring. Figure 5 As shown, on day 0, equal amounts of water were added to sample bottles from different groups. The rate of water loss in the bottles was recorded daily, and the water loss curve was recorded. It can be seen that the Janus membrane dressing with a double-layer structure significantly reduced water loss compared to the single-layer Gelatin (Gelatin Only), and maintained a high water level even after 21 days.

[0089] Example of effect 4

[0090] The mechanical properties of the Janus-structured bilayer membrane, the single-layer Gelatin (Gelatin Only) membrane, and the single-layer PTMC (PTMC Only) membrane obtained in the comparative examples were compared. Membrane materials were cut into regularly shaped rectangular strips (60 mm long, 5 mm wide, and 0.15 mm thick). To more closely approximate actual usage, the samples were first wetted, and then the stress-strain curves of the different membrane materials were tensile tested using a universal testing machine. The results are shown below. Figure 6 As shown:

[0091] Compared to gelatin-only films, PTMC-only films exhibit significantly greater toughness. For example... Figure 6 As shown in Figure a, PTMCOnly films can be stretched to a maximum length of nearly 200%, while Gelatin Only films fracture when stretched to approximately 37% strain. Figure 6 As shown in Figure c, the Janus membrane formed by combining the two layers exhibits significantly improved toughness, as evidenced by a marked increase in elongation at break, rising from approximately 36.7% for a single-layer gelatin-only membrane to around 70.6%. This result demonstrates that combining the PTMC layer with the gelatin layer effectively toughens the gelatin material, overcoming the poor mechanical properties of traditional gelatin hydrogels and broadening its application in wound repair.

[0092] Example 5

[0093] This study compared the blocking abilities of Janus-structured bilayer membranes and single-layer Gelatin (Gelatin Only) against typical Gram-negative bacteria *Escherichia coli* (E. coli) and Gram-positive bacteria *Staphylococcus aureus* (S. aureus). Gelatin and Janus membranes were cut into discs approximately 10 mm in diameter, and a concentration of 5 × 10⁻⁶ was used. 8 The bacterial suspension at a concentration of CFU / ml was diluted with LB medium and placed in an autoclaved sample bottle (bacterial suspension bottle). Discs of different membrane materials were attached to the bottle neck. The sample bottle was inverted, and another autoclaved sample bottle was attached underneath as a collection bottle. Figure 8 As shown in a. After standing at room temperature for 7 days, the device was opened, LB medium was added to the collection bottle, and the bacteria on the membrane side that were not in direct contact with the bacterial solution were gently scraped. The plate was diluted and the number of colonies was calculated to evaluate the isolation effect of different membrane materials on bacteria. The residual ratio = colony concentration of bacterial solution on the plate / initial concentration of bacterial solution * 100%.

[0094] like Figure 8 As shown in b, for Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), Janus membranes with a dense PTMC outer layer effectively reduce the permeability of both bacteria compared to Gelatin Only membranes. Figure 7 At dilution ratio b, the number of Escherichia coli and Staphylococcus aureus colonies was significantly reduced, demonstrating a high bacterial isolation effect. Figure 8 As shown in Figure c, the residual E. coli colonies on a gelatin-only membrane are approximately 12%, while using a double-layer membrane can reduce this to 0.2%. This is because Staphylococcus aureus has a smaller average particle size. Figure 8 While Staphylococcus aureus may have stronger permeability to different membranes, the bilayer membrane still showed a significant barrier effect, with a residual rate of only 0.16%, compared to the 23% bacterial residue rate of the Gelatin Only membrane. Therefore, after local wound debridement, the biomimetic bilayer membrane with a Janus structure can effectively reduce the adhesion and infection of external pathogens in the wound without the need for other additives to reduce wound irritation.

[0095] Mesenchymal stem cells (MSCs) were cultured in culture media containing different concentrations of Janus bilayer membrane extract (0 mg / ml, 5 mg / ml, 10 mg / ml). Cell viability in different experimental groups at days 1, 4, and 7 was detected using the CCK-8 assay to determine whether the material exhibited significant cytotoxicity. Figure 8The results showed that no significant cell death was observed in MSCs cultured in any concentration of medium on Days 1 and 4. Although the high concentration (10 mg / ml) group showed a certain decrease in cell viability in the Day 7 test results, the concentration used in actual applications is far less than 10 mg / ml.

[0096] Example 6

[0097] To verify the practical application effect of the yin-yang double-layer film dressing prepared in this invention, the dressing was used to repair the wound of a mouse total skin excision model.

[0098] In 6-week-old ICR mice, a total skin excision was performed on the back to create a wound model, with a wound diameter of approximately 1 cm. Gelatin Only dressing, Janus dressing (Janus group), and Janus dressing loaded with exosomes derived from umbilical cord mesenchymal stem cells (Janus-exo group) were cut into small round pieces with a diameter of approximately 1 cm and applied to the back of the mice to promote wound healing. The effects of different treatment groups on promoting wound repair were compared. Wound closure was recorded by photographing on Day 0, Day 4, Day 7, Day 10, and Day 15. The results are as follows: Figure 9 As shown. (Wound closure rate = Day × Wound area size / Day 0 Wound area size * 100%)

[0099] like Figure 9As shown, the wound closure rates of different treatment groups were recorded on Day 4, Day 7, Day 10, and Day 15. On Day 4, the Janus-exo group showed excellent wound closure, reaching approximately 50% by Day 4 alone, indicating the significant regulatory effect of exosomes on the local wound microenvironment. Meanwhile, the double-layer Janus dressing treatment group and the Gelatin Only treatment group showed better results than the Control group. On Day 7, while the Janus-exo group maintained excellent wound healing, the Janus group also showed a better wound closure rate. This is presumably because the Janus dressing's ability to keep the wound moist over time positively impacted wound healing, promoting local cell migration and granulation tissue regeneration. However, the Gelatin Only group, due to the rapid drying of the gelatin monolayer, did not show a significant promoting effect on wound healing. On Day 10, the wounds in all groups further shrank. At this point, the wounds in the Janus-exo group tended to be completely closed, and significant hair regrowth was observed in the wound area, a positive signal of complete skin repair. At this point, the Janus group showed significantly better repair than the Gelatin Only group. On Day 15, the wounds in both the Janus-exo and Janus dressing groups tended to be completely closed, and good hair follicle regeneration was observed. The Control and Gelatin Only groups, however, still required some time to achieve complete wound repair.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A wound dressing with a yin-yang structure, characterized in that, It consists of two layers: the upper layer is a dense hydrophobic layer, which is a polytrimethylene carbonate fiber membrane with a biomimetic epidermal structure; the lower layer is a hydrophilic layer, which is a natural polymer fiber hydrogel membrane that simulates the structure of the dermis. The polytrimethylene carbonate fiber membrane is a combination of a photocrosslinkable polytrimethylene carbonate fiber membrane and a high molecular weight polytrimethylene carbonate fiber membrane, wherein the synthesis method of the photocrosslinkable polytrimethylene carbonate fiber membrane is as follows: Trimethylolpropane and stannous octoate were used as initiator and catalyst, respectively, to initiate the ring-opening polymerization of trimethylene carbonate monomers at 140°C, yielding three-armed polytrimethylene carbonate oligomers. Subsequently, a chemical reaction was carried out between methacrylic anhydride and the polytrimethylene carbonate fiber membrane oligomers to ultimately obtain photocrosslinkable polytrimethylene carbonate fiber membranes. The molecular weight of the synthesized photocrosslinkable polytrimethylene carbonate fiber membranes was controlled at 10–50 kDa, with a grafting rate of 50–80%. The method for synthesizing the high molecular weight PTMC is as follows: High molecular weight PTMC was synthesized by bulk polymerization of trimethylene carbonate, with a molecular weight of 100~500 kDa.

2. The yin-yang structured wound dressing according to claim 1, characterized in that, The hydrophobic layer, a polytrimethylene carbonate fiber membrane, has a thickness of 10-400 μm, a fiber diameter distribution of 0.5-2 μm, and a pore size of ≤1 μm; the hydrophilic layer, a natural polymer fiber hydrogel membrane, has a thickness of 20-500 μm, a fiber diameter mainly distributed in the range of 0.1-2 μm, and a pore size of 10-150 μm.

3. The yin-yang structured wound dressing according to claim 1, characterized in that, The material of the natural polymer fiber hydrogel membrane is at least one of gelatin, collagen and silk fibroin, or a composite material formed by mixing two or three of gelatin, collagen and silk fibroin, or a composite material formed by mixing gelatin, collagen or silk fibroin with other natural polymer materials.

4. The yin-yang structured wound dressing according to claim 3, characterized in that, The other natural polymer materials mentioned are chitosan, hyaluronic acid, or sodium alginate.

5. A method for preparing a multifunctional wound dressing with a yin-yang membrane structure, characterized in that, Includes the following steps: (1) Preparation of fiber hydrogel membrane with hydrophilic layer A photocrosslinkable natural polymer material is formulated into a spinning solution, a photoinitiator is added to the spinning solution, and a fiber hydrogel membrane is prepared by electrospinning technology. (2) Electrospinning the hydrophobic layer of fibers onto the hydrophilic layer of fiber hydrogel membrane. Photocrosslinkable polytrimethylene carbonate and high molecular weight polytrimethylene carbonate are mixed to prepare a spinning solution. A photoinitiator is added to the spinning solution, and the spinning solution is sprayed onto the fiber hydrogel membrane prepared in step (1) by electrospinning technology to obtain a bilayer membrane with hydrophobic and hydrophilic properties. (3) Photocrosslinked bilayer film Prepare an ethanol solution containing a photoinitiator as a crosslinking solution. Immerse the bilayer film prepared in step (2) completely in the crosslinking solution and perform ultraviolet crosslinking. After the crosslinking is completed, remove the material from the crosslinking solution, rinse the film material with ethanol to remove the residual photoinitiator, then rinse with deionized water to remove the ethanol and freeze dry to obtain the final product. Synthesis of the photocrosslinkable polytrimethylene carbonate: Trimethylolpropane and stannous octoate were used as initiator and catalyst, respectively, to initiate the ring-opening polymerization of trimethylene carbonate monomers at a high temperature of 140°C, yielding three-armed polytrimethylene carbonate oligomers. Subsequently, methacrylic anhydride and the polytrimethylene carbonate oligomers underwent a chemical reaction to ultimately obtain photocrosslinkable polytrimethylene carbonate. The molecular weight of the synthesized photocrosslinkable polytrimethylene carbonate was controlled at 10–50 kDa, with a grafting rate of 50–80%. The high molecular weight polytrimethylene carbonate was synthesized as follows: High molecular weight polytrimethylene carbonate was synthesized by bulk polymerization of trimethylene carbonate, and the molecular weight of the synthesized high molecular weight polytrimethylene carbonate was 100~500 kDa.

6. The method for preparing the multifunctional wound dressing with the yin-yang membrane structure according to claim 5, characterized in that, In step (1): The photoinitiator has a mass fraction of 0.1% to 5% relative to the photocrosslinkable natural polymer material; The thickness of the fiber hydrogel membrane is controlled between 20 and 500 μm.

7. The method for preparing the multifunctional wound dressing with the yin-yang membrane structure according to claim 5, characterized in that, The synthesis of the photocrosslinkable natural polymer material is achieved by reacting methacrylic anhydride with a natural polymer material, with the grafting degree controlled within the range of 30% to 95%.

8. The method for preparing the multifunctional wound dressing with the yin-yang membrane structure according to claim 5, characterized in that, In step (2): The mass ratio of photocrosslinkable PTMC to high molecular weight polytrimethylene carbonate is 1%~10%; The photoinitiator has a mass fraction of 0.1% to 5% relative to the spinning solution.

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