A wound dressing for chronic wounds and a method of making the same

Abstract

The present application relates to the technical field of wound repair material, in particular to a chronic wound dressing and a preparation method thereof, the wound dressing is prepared from a first contact layer, an intermediate transition layer and a backing layer which are in direct contact with the wound, through the unique three-layer structure design, a chronic wound dressing with fast healing speed, strong anti-inflammatory and antibacterial properties, high liquid absorption and effective regulation of the microenvironment of the wound is prepared, the first contact layer is in direct contact with the wound, absorbs the exudate, plays an anti-inflammatory and antioxidant role, the intermediate transition layer guides the exudate from the first contact layer to the backing layer through the asymmetric fiber membrane structure on both sides and does not back permeate, the backing layer has a connected microporous structure, has strong air permeability and can absorb a large amount of exudate, provides a humid environment for the wound, inhibits bacterial growth and promotes wound healing, compared with the prior art, has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of wound repair materials, and in particular to a wound dressing for chronic wounds and its preparation method. Background Technology

[0002] Chronic wounds generally refer to wounds caused by various reasons that have failed to heal or show no tendency to heal after more than one month of regular treatment. Chronic, difficult-to-heal wounds on the body surface often occur in patients with severe chronic diseases such as diabetes, trauma, varicose veins, arteriosclerosis, and paraplegia requiring prolonged bed rest, as well as patients with acute injuries. They are characterized by complex pathogenesis, high treatment difficulty, long treatment cycles, and high costs. With population aging and changes in disease patterns, the number of patients with chronic, difficult-to-heal wounds on the body surface caused by various diseases, in addition to acute wounds caused by trauma and burns, is increasing year by year. Normal wound healing consists of four stages: hemostasis, inflammation, proliferation, and remodeling. However, due to reduced release of anti-inflammatory factors and bacterial / fungal infections in most patients with chronic wounds, wound repair remains stuck in the inflammatory response stage, making it difficult to transition to the next stage. This results in chronic, difficult-to-heal wounds on the body surface healing slower than normal wounds. Currently, the main treatment methods for chronic, difficult-to-heal wounds on the body surface include drug therapy, physical therapy, surgical debridement, and wound dressings—modern medical methods. Despite the proliferation of methods, the treatment effects remain less than ideal.

[0003] Dressings play a vital role in wound protection and treatment. Traditional dressings such as gauze and bandages are simple to use and inexpensive, but their function is limited. Furthermore, the large pore size of gauze cannot effectively prevent infection caused by bacterial infiltration. During wound repair, granulation tissue can adhere to the mesh, causing pulling during dressing changes, resulting in pain for the patient and delaying the healing process. Therefore, traditional dressings are less effective for chronic wounds with complex tissue conditions.

[0004] In the prior art, such as patent document CN119499439B, a method for preparing and applying a pH / glucose dual-response chronic wound dressing containing frankincense essential oil is disclosed, including: (1) preparing a hydrogel that can fill skin surface wounds and perform dual dynamic covalent cross-linking adhesiveness by using 4-formaldehyde phenylboronic acid as a cross-linking agent to form Schiff base and borate ester bonds with carboxymethyl chitosan and two different molecular weight hyaluronic acids, respectively, and freeze-drying it for later use; (2) preparing frankincense essential oil into microcapsules by emulsification and coagulation method. , freeze-dry for later use; (3) mix the hydrogel with the freeze-dried powder of frankincense oil microcapsules, add deionized water or PBS and stir to reconstitute to form a pH / glucose dual-response hybrid hydrogel dressing coated with frankincense oil microcapsules; this new type of wound dressing has significant improvements in moisturizing and healing compared with traditional gauze, and has anti-inflammatory, antioxidant, antibacterial and healing-promoting effects. However, the saturated single gel dressing cannot drain excess exudate, which causes the wound to be in a moist environment for a long time, which is easy to breed bacteria and thus affect the healing of the wound.

[0005] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop a wound dressing for chronic wounds and its preparation method. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a wound dressing for chronic wounds and its preparation method, so as to solve the problems of difficult drainage of exudate and persistent inflammatory response in existing wound dressings.

[0007] To achieve the above objectives, the present invention provides a wound dressing for chronic wounds and a method for preparing the same.

[0008] A wound dressing for chronic wounds, the wound dressing being prepared from a first contact layer, an intermediate transition layer, and a backing layer that are in direct contact with the wound; The first contact layer was prepared from methacrylamide gelatin, oxidized angelica polysaccharide and hirudin liposomes; The intermediate transition layer is a fiber membrane with asymmetrical pore sizes on both sides, with the side with larger pore size attached to the first contact layer and the side with smaller pore size attached to the backing layer. The backing layer is prepared from polyvinyl alcohol, sodium alginate and Bifidobacterium bifidum.

[0009] Preferably, the method for preparing the hirudin liposomes is as follows: Step A1. Add lecithin and cholesterol to chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 40-42℃ to remove the organic solvent until a uniform film is formed in the eggplant flask. Step A2. Add hirudin to a phosphate buffer solution with a pH of 7.2-7.4, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle into a flask and hydrate it at 35-38℃ and 200-260 rpm for 30-40 minutes. After hydration, perform ultrasonication in an ice bath using an ultrasonic cell disruptor to obtain hirudin liposomes.

[0010] Preferably, the ratio of lecithin, cholesterol and chloroform used in step A1 is 100-150mg:60-90mg:50-90mL; The concentration of the phosphate buffer solution mentioned in step A2 is 0.02 mmol / L; The ratio of phosphate buffer solution to hirudin is 10-20 mL: 1-2 mg.

[0011] Preferably, the first contact layer is prepared by the following method: Step B1. Add gelatin to a 0.02 mmol / L phosphate buffer solution with a pH of 7.2-7.4, and add methylpropyl anhydride dropwise while stirring. Stir at 600-800 rpm for 2-4 hours. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. Step B2. Add sodium periodate to a 2% aqueous solution of Angelica polysaccharide and stir in the dark at 37-42℃ for 10-12 hours. After stirring, add ethylene glycol to stop the reaction, then add 95% ethanol and let stand at 0-4℃ for 18-20 hours. Centrifuge and freeze dry to obtain oxidized Angelica polysaccharide. Step B3. Add methacrylated gelatin to a phosphate buffer solution, sonicate for 3-5 minutes, then add oxidized angelica polysaccharide, hirudin liposomes and phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer.

[0012] Preferably, the ratio of gelatin, phosphate buffer solution and methylpropionic anhydride used in step B1 is 1-1.5g:10-20mL:0.7-1.2mL; The ratio of sodium periodate, Angelica polysaccharide aqueous solution, ethylene glycol and ethanol used in step B2 is 0.6-1g:50-100mL:75-150mL:500-2000mL; The mass ratio of methacrylated gelatin, phosphate buffer solution, oxidized Angelica polysaccharide, hirudin liposomes and phenyl-2,4,6-trimethylbenzoylphosphonic acid in step B3 is 10-18:100-200:3-6:3-5.5:0.1-0.15.

[0013] Preferably, the intermediate transition layer is prepared by the following method: Step C1. Add chitosan to a 50% acetic acid aqueous solution and stir at room temperature for 12-18 hours to obtain mixture B; then add polyethylene oxide to a 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add volumes V1 and V2 of mixture C to 10 mL of mixture B and stir for 3-5 hours. After standing for 18-24 hours, centrifuge to obtain spinning solution A and spinning solution B, respectively. Step C2. Using the dual-channel electrospinning method, add spinning solution A and spinning solution B to two 10mL syringes respectively, connect the electrospinning machine, set the electrospinning parameters, and perform electrospinning to obtain the intermediate transition layer.

[0014] Preferably, the ratio of chitosan to acetic acid aqueous solution in step C1 is 3-5g:100mL; The ratio of the amount of polyethylene oxide to the aqueous acetic acid solution is 2-3 g: 100 mL; The value of V1 is 6.5-7.9 mL; V2 is 10 mL; In step C2, the electrospinning parameters are: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 2-3h.

[0015] Preferably, the backing layer is prepared by the following method: A 10% (w / w) aqueous solution of polyvinyl alcohol and a 2% (w / w) aqueous solution of sodium alginate were mixed and stirred at 37-42℃ for 2-3 hours. After cooling to room temperature, a suspension of Bifidobacterium bifidum was added, mixed evenly, and allowed to stand for 2-3 hours to remove air bubbles. The mixture was then poured into a mold, freeze-dried, and thawed at room temperature for 4 hours. This process was repeated for 3 cycles and freeze-dried again to obtain the backing layer.

[0016] Preferably, the volume ratio of the polyvinyl alcohol aqueous solution, sodium alginate aqueous solution, and Bifidobacterium bifidum suspension is 20-30:20-35:3-5; The freeze-drying process is carried out at a temperature of -20 to 17°C for 18 to 20 hours.

[0017] A method for preparing a wound dressing for chronic wounds includes the following steps: The first contact layer is attached to the side of the intermediate transition layer with larger pore size and photocured by irradiation with ultraviolet light at a wavelength of 365nm. Then, the backing layer is attached to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

[0018] The beneficial effects of this invention are: This invention provides a wound dressing for chronic wounds and its preparation method. The wound dressing of this invention is prepared from a first contact layer, an intermediate transition layer, and a backing layer that directly contact the wound. Through a unique three-layer structure design, a wound dressing for chronic wounds with fast healing speed, strong anti-inflammatory and antibacterial properties, high exudate absorption, and the ability to effectively regulate the wound microenvironment is obtained. The first contact layer directly contacts the wound, absorbs exudate, and exerts anti-inflammatory and antioxidant effects. The intermediate transition layer guides exudate from the first contact layer to the backing layer through a fiber membrane structure with asymmetrical pore sizes on both sides, preventing backflow. The backing layer has a connected microporous structure, which has high air permeability and can also absorb a large amount of exudate, providing a moist environment for the wound, inhibiting bacterial growth, and promoting wound healing. Compared with the prior art, it has broad application prospects. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0020] The Bifidobacterium bifidum used in this invention is Bifidobacterium bifidum BD-1, which is disclosed in the invention patent with authorization announcement number "CN117752696A" and title "Application of Bifidobacterium bifidum BD-1 and / or its metabolites", and accession number CGMCCNO.24517.

[0021] Example 1: A method for preparing a wound dressing for chronic wounds, comprising the following steps: S1. Inoculate Bifidobacterium bifidum BD-1 into TOS solid medium and anaerobically culture at 37°C. After stabilization, scrape off the entire colony and measure the OD600 value of the bacterial solution using a spectrophotometer. Adjust the bacterial concentration to 2×10⁻⁶ using PBS buffer solution. 8 The cfu / ml was then inoculated into 3ml of TPY liquid medium and anaerobic cultured at 37℃ for 48h. After centrifugation at 12000r / min for 3min, the supernatant was discarded, the bacterial cells were collected, and a bacterial suspension was prepared with 30mL of sterile water. S2. Add 100mg of lecithin and 60mg of cholesterol to 50mL of chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 40℃ to remove the organic solvent until a uniform film is formed in the eggplant flask. S3. Add 1 mg of hirudin to 10 mL of phosphate buffer solution with pH 7.2, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle to a flask and hydrate it at 35 °C and 200 rpm for 30 min. After hydration, perform ice bath ultrasonication in an ultrasonic cell disruptor to obtain hirudin liposomes. S4. Add 1g of gelatin to 10mL of phosphate buffer solution with pH 7.2, and add 0.7mL of methylpropionic anhydride dropwise while stirring. Stir at 600rpm for 2h. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. S5. Add 0.6g of sodium periodate to 50mL of 2% Angelica polysaccharide aqueous solution, stir at 37℃ in the dark for 10h, after stirring, add 75mL of ethylene glycol to stop the reaction, then add 500mL of 95% ethanol, let stand at 0℃ for 18h, centrifuge, freeze dry to obtain oxidized Angelica polysaccharide. S6. Add 10g of methacrylated gelatin to 100g of phosphate buffer solution, sonicate for 3min, then add 3g of oxidized angelica polysaccharide, 3g of hirudin liposomes and 0.1g of phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer; S7. Add 3g of chitosan to 100mL of 50% acetic acid aqueous solution and stir at room temperature for 12h to obtain mixture B; then add 2g of polyethylene oxide to 100mL of 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add 6.5mL and 10mL of mixture C to 10mL of mixture B and stir for 3h. After standing for 18h, centrifuge to obtain spinning solution A and spinning solution B respectively. S8. Using a dual-channel electrospinning method, spinning solution A and spinning solution B were added to two 10mL syringes respectively. The electrospinning machine was connected and the electrospinning parameters were set as follows: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 2h. Electrospinning was performed to obtain an intermediate transition layer. S9. Mix 20 mL of 10% polyvinyl alcohol aqueous solution with 20 mL of 2% sodium alginate aqueous solution, stir at 37℃ for 2 h, cool to room temperature, add 3 mL of Bifidobacterium bifidum suspension, mix evenly, let stand for 2 h to remove air bubbles, pour into a mold, freeze dry at -17℃ for 18 h, thaw at room temperature for 4 h, repeat 3 cycles and freeze dry again to obtain the backing layer; S10. Attach the first contact layer to the side of the intermediate transition layer with larger pore size and cure it with ultraviolet light with a wavelength of 365nm. Then attach the backing layer to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

[0022] Example 2: A method for preparing a wound dressing for chronic wounds, comprising the following steps: S1. Inoculate Bifidobacterium bifidum BD-1 into TOS solid medium and anaerobically culture at 37°C. After stabilization, scrape off the entire colony and measure the OD600 value of the bacterial solution using a spectrophotometer. Adjust the bacterial concentration to 2×10⁻⁶ using PBS buffer solution. 8 The cfu / ml was then inoculated into 3ml of TPY liquid medium and anaerobic cultured at 37℃ for 48h. After centrifugation at 12000r / min for 3min, the supernatant was discarded, the bacterial cells were collected, and a bacterial suspension was prepared with 30mL of sterile water. S2. Add 120 mg of lecithin and 70 mg of cholesterol to 65 mL of chloroform, disperse evenly by ultrasonication, and then remove the organic solvent by rotary evaporation at 41 °C using a rotary evaporator until a uniform film is formed in the eggplant flask. S3. Add 1.4 mg of hirudin to 13 mL of phosphate buffer solution with pH 7.3, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle to a flask and hydrate it at 36 °C and 220 rpm for 35 min. After hydration, perform ultrasonication in an ice bath in an ultrasonic cell disruptor to obtain hirudin liposomes. S4. Add 1.2g of gelatin to 14mL of phosphate buffer solution with pH 7.3, and add 0.9mL of methylpropionic anhydride dropwise while stirring. Stir at 700rpm for 3h. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. S5. Add 0.7g sodium periodate to 70mL of 2% Angelica polysaccharide aqueous solution, stir at 39℃ in the dark for 11h, after stirring, add 100mL ethylene glycol to stop the reaction, then add 1000mL 95% ethanol, let stand at 2℃ for 19h, centrifuge, freeze dry to obtain oxidized Angelica polysaccharide; S6. Add 12.4g of methacrylated gelatin to 130g of phosphate buffer solution, sonicate for 3min, then add 4g of oxidized angelica polysaccharide, 4g of hirudin liposomes and 0.12g of phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer; S7. Add 3.5g of chitosan to 100mL of 50% acetic acid aqueous solution and stir at room temperature for 14h to obtain mixture B; then add 2.4g of polyethylene oxide to 100mL of 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add 6.9mL and 10mL of mixture C to 10mL of mixture B and stir for 4h. After standing for 20h, centrifuge to obtain spinning solution A and spinning solution B respectively. S8. Using a dual-channel electrospinning method, spinning solution A and spinning solution B were added to two 10mL syringes respectively. The syringes were connected to an electrospinning machine, and the electrospinning parameters were set as follows: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 2.5h. Electrospinning was then performed to obtain an intermediate transition layer. S9. Mix 25 mL of 10% polyvinyl alcohol aqueous solution with 25 mL of 2% sodium alginate aqueous solution, stir at 40℃ for 2.5 h, cool to room temperature, add 4 mL of Bifidobacterium bifidum suspension, mix evenly, let stand for 2.5 h to remove air bubbles, pour into a mold, freeze dry at -18℃ for 19 h, thaw at room temperature for 4 h, repeat 3 cycles and freeze dry again to obtain the backing layer; S10. Attach the first contact layer to the side of the intermediate transition layer with larger pore size and cure it with ultraviolet light with a wavelength of 365nm. Then attach the backing layer to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

[0023] Example 3: A method for preparing a wound dressing for chronic wounds, comprising the following steps: S1. Inoculate Bifidobacterium bifidum BD-1 into TOS solid medium and anaerobically culture at 37°C. After stabilization, scrape off the entire colony and measure the OD600 value of the bacterial solution using a spectrophotometer. Adjust the bacterial concentration to 2×10⁻⁶ using PBS buffer solution. 8 The cfu / ml was then inoculated into 3ml of TPY liquid medium and anaerobic cultured at 37℃ for 48h. After centrifugation at 12000r / min for 3min, the supernatant was discarded, the bacterial cells were collected, and a bacterial suspension was prepared with 30mL of sterile water. S2. Add 140mg of lecithin and 80mg of cholesterol to 80mL of chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 42℃ to remove the organic solvent until a uniform film is formed in the eggplant flask. S3. Add 1.7 mg of hirudin to 17 mL of phosphate buffer solution with pH 7.4, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle to a flask and hydrate it at 38 °C and 240 rpm for 38 min. After hydration, perform ultrasonication in an ultrasonic cell disruptor in an ice bath to obtain hirudin liposomes. S4. Add 1.4g of gelatin to 18mL of phosphate buffer solution with pH 7.4, and add 1mL of methylpropionic anhydride dropwise while stirring. Stir at 800rpm for 4h. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. S5. Add 0.9g of sodium periodate to 80mL of 2% Angelica polysaccharide aqueous solution, stir at 42℃ in the dark for 12h, after stirring, add 120mL of ethylene glycol to stop the reaction, then add 1500mL of 95% ethanol, let stand at 4℃ for 18h, centrifuge, freeze dry to obtain oxidized Angelica polysaccharide; S6. Add 15g of methacrylated gelatin to 170g of phosphate buffer solution, sonicate for 4min, then add 5g of oxidized angelica polysaccharide, 5g of hirudin liposomes and 0.13g of phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer; S7. Add 4.5g of chitosan to 100mL of 50% acetic acid aqueous solution and stir at room temperature for 16h to obtain mixture B; then add 2.6g of polyethylene oxide to 100mL of 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add 7.3mL and 10mL of mixture C to 10mL of mixture B and stir for 4.5h. After standing for 22h, centrifuge to obtain spinning solution A and spinning solution B, respectively. S8. Using a dual-channel electrospinning method, spinning solution A and spinning solution B were added to two 10mL syringes respectively. The electrospinning machine was connected and the electrospinning parameters were set as follows: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 3h. Electrospinning was performed to obtain an intermediate transition layer. S9. Mix 27 mL of 10% polyvinyl alcohol aqueous solution with 30 mL of 2% sodium alginate aqueous solution, stir at 37℃ for 3 h, cool to room temperature, add 4.5 mL of Bifidobacterium bifidum suspension, mix evenly, let stand for 3 h to remove air bubbles, pour into a mold, freeze dry at -18℃ for 20 h, thaw at room temperature for 4 h, repeat 3 cycles and freeze dry again to obtain the backing layer; S10. Attach the first contact layer to the side of the intermediate transition layer with larger pore size and cure it with ultraviolet light with a wavelength of 365nm. Then attach the backing layer to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

[0024] Example 4: A method for preparing a wound dressing for chronic wounds, comprising the following steps: S1. Inoculate Bifidobacterium bifidum BD-1 into TOS solid medium and anaerobically culture at 37°C. After stabilization, scrape off the entire colony and measure the OD600 value of the bacterial solution using a spectrophotometer. Adjust the bacterial concentration to 2×10⁻⁶ using PBS buffer solution. 8The cfu / ml was then inoculated into 3ml of TPY liquid medium and anaerobic cultured at 37℃ for 48h. After centrifugation at 12000r / min for 3min, the supernatant was discarded, the bacterial cells were collected, and a bacterial suspension was prepared with 30mL of sterile water. S2. Add 150 mg of lecithin and 90 mg of cholesterol to 90 mL of chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 42 °C to remove the organic solvent until a uniform film is formed in the eggplant flask. S3. Add 2 mg of hirudin to 20 mL of phosphate buffer solution with pH 7.4, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle to a flask and hydrate it at 38 °C and 260 rpm for 40 min. After hydration, perform ice bath ultrasonication in an ultrasonic cell disruptor to obtain hirudin liposomes. S4. Add 1.5g of gelatin to 20mL of phosphate buffer solution with pH 7.4, and add 1.2mL of methylpropionic anhydride dropwise while stirring. Stir at 800rpm for 4h. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. S5. Add 1g of sodium periodate to 100mL of 2% Angelica polysaccharide aqueous solution, stir at 42℃ in the dark for 12h, after stirring, add 150mL of ethylene glycol to stop the reaction, then add 2000mL of 95% ethanol, let stand at 4℃ for 20h, centrifuge, freeze dry to obtain oxidized Angelica polysaccharide. S6. Add 18g of methacrylated gelatin to 200g of phosphate buffer solution, sonicate for 5min, then add 6g of oxidized angelica polysaccharide, 5.5g of hirudin liposomes and 0.15g of phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer; S7. Add 5g of chitosan to 100mL of 50% acetic acid aqueous solution and stir at room temperature for 18h to obtain mixture B; then add 3g of polyethylene oxide to 100mL of 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add 7.9mL and 10mL of mixture C to 10mL of mixture B and stir for 5h. After standing for 24h, centrifuge to obtain spinning solution A and spinning solution B respectively. S8. Using a dual-channel electrospinning method, spinning solution A and spinning solution B were added to two 10mL syringes respectively. The electrospinning machine was connected and the electrospinning parameters were set as follows: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 3h. Electrospinning was performed to obtain an intermediate transition layer. S9. Mix 30 mL of 10% polyvinyl alcohol aqueous solution with 35 mL of 2% sodium alginate aqueous solution, stir at 42℃ for 3 h, cool to room temperature, add 5 mL of Bifidobacterium bifidum suspension, mix evenly, let stand for 3 h to remove air bubbles, pour into a mold, freeze dry at -20℃ for 20 h, thaw at room temperature for 4 h, repeat 3 cycles and freeze dry again to obtain the backing layer; S10. Attach the first contact layer to the side of the intermediate transition layer with the larger pore size, and then use ultraviolet light with a wavelength of 365nm for photocuring. Attach the backing layer to the side of the intermediate transition layer with the smaller pore size to obtain the wound dressing for chronic wounds.

[0025] Comparative Example 1: Compared with Example 1, this comparative example did not add hirudin liposomes during the preparation of the first contact layer. All other steps and parameters were the same, and will not be repeated here. The final wound dressing was obtained.

[0026] Comparative Example 2: Compared with Example 1, this comparative example did not add Bifidobacterium bifidum suspension during the preparation of the backing layer. All other steps and parameters were the same, and will not be repeated here. The final wound dressing was obtained.

[0027] Comparative Example 3: This comparative example differs from Example 1 only in that "hirudin liposomes" is replaced with "hirudin". All other steps and parameters are the same, and will not be repeated here. The final result is a bone repair hydrogel.

[0028] Comparative Example 4: S1. Inoculate Bifidobacterium bifidum BD-1 into TOS solid medium and anaerobically culture at 37°C. After stabilization, scrape off the entire colony and measure the OD600 value of the bacterial solution using a spectrophotometer. Adjust the bacterial concentration to 2×10⁻⁶ using PBS buffer solution. 8 The cfu / ml was then inoculated into 3ml of TPY liquid medium and anaerobic cultured at 37℃ for 48h. After centrifugation at 12000r / min for 3min, the supernatant was discarded, the bacterial cells were collected, and a bacterial suspension was prepared with 30mL of sterile water. S2. Add 100mg of lecithin and 60mg of cholesterol to 50mL of chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 40℃ to remove the organic solvent until a uniform film is formed in the eggplant flask. S3. Add 1 mg of hirudin to 10 mL of phosphate buffer solution with pH 7.2, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle to a flask and hydrate it at 35 °C and 200 rpm for 30 min. After hydration, perform ice bath ultrasonication in an ultrasonic cell disruptor to obtain hirudin liposomes. S4. Add 1g of gelatin to 10mL of phosphate buffer solution with pH 7.2, and add 0.7mL of methylpropionic anhydride dropwise while stirring. Stir at 600rpm for 2h. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. S5. Add 0.6g of sodium periodate to 50mL of 2% Angelica polysaccharide aqueous solution, stir at 37℃ in the dark for 10h, after stirring, add 75mL of ethylene glycol to stop the reaction, then add 500mL of 95% ethanol, let stand at 0℃ for 18h, centrifuge, freeze dry to obtain oxidized Angelica polysaccharide. S6. Add 10g of methacrylated gelatin to 100g of phosphate buffer solution, sonicate for 3min, then add 3g of oxidized angelica polysaccharide, 3g of hirudin liposomes and 0.1g of phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer; S7. Add 3g of chitosan to 100mL of 50% acetic acid aqueous solution and stir at room temperature for 12h to obtain mixture B; then add 2g of polyethylene oxide to 100mL of 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add 10mL of mixture C to 10mL of mixture B and mix and stir for 3h. After standing for 18h, centrifuge to obtain spinning solution. S8. Electrospinning was performed using the following parameters: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 2h. This process yielded an intermediate transition layer. S9. Mix 20 mL of 10% polyvinyl alcohol aqueous solution with 20 mL of 2% sodium alginate aqueous solution, stir at 37℃ for 2 h, cool to room temperature, add 3 mL of Bifidobacterium bifidum suspension, mix evenly, let stand for 2 h to remove air bubbles, pour into a mold, freeze dry at -17℃ for 18 h, thaw at room temperature for 4 h, repeat 3 cycles and freeze dry again to obtain the backing layer; S10. Attach the first contact layer to the side of the intermediate transition layer with larger pore size and cure it with ultraviolet light with a wavelength of 365nm. Then attach the backing layer to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

[0029] Performance testing: Adhesion strength - tensile test: The wound dressings prepared in Examples 1-4 and Comparative Examples 1-4 were cut into 2cm × 2cm squares. One side was adhered to moist pigskin, and the other side was connected to a digital tensile tester. The entire testing device was immersed in PBS solution, and a tensile tester was used to apply a tensile force at 10mm / min until the adhesive interface separated. The tensile force value at separation was recorded, and the adhesive strength (Pa) was calculated based on the adhesive area. Water vapor transmission rate test: The water vapor transmission rate (WVTR) of chronic wound repair materials was determined according to the ASTM E96-00 method of the American Bureau of Standards. The specific steps are as follows: First, 10 mL of deionized water was placed in a 13 mm diameter vial. Then, the wound dressings prepared in Examples 1-4 and Comparative Examples 1-4 were cut into 1.5 cm × 1.5 cm pieces and placed at the mouth of the vial. The chronic wound repair material was then tightly fixed to the mouth of the vial using sealant and weighed. The sample vial was then placed in a constant temperature and humidity incubator (temperature 37°C, relative humidity 79%) for 24 hours. Afterward, the vial was weighed. Formula for calculating water vapor transmission rate: Water vapor transmission rate (WVTR) =

[0030] in, t represents the weight of water loss over 24 hours (g / 24h), and A represents the surface area of ​​the bottle opening (mm²). 2 ); Water absorption rate test: The wound dressings prepared in Examples 1-4 and Comparative Examples 1-4 were cut into 2cm×2cm squares, weighed, and denoted as Mo. The chronic wound repair material was then placed in PBS solution for 30 minutes. The sample was then removed from the PBS and the surface moisture was quickly absorbed with absorbent paper. The sample mass was weighed and denoted as Mw. Water absorption rate calculation formula: Water absorption rate (%) = ×100% Free radical scavenging rate test: The free radical scavenging rate of the test material (DPPH free radical scavenging rate detection experiment) was used to demonstrate its antioxidant effect. The test method was as follows: 10 mg of the wound dressing samples prepared in Examples 1-4 and Comparative Examples 1-4 were cut and soaked in 1 mL of PBS at 37°C for 1 h. After soaking, the samples were removed and gently squeezed to obtain the liquid as the test solution. 200 μL of the test solution was taken and 3.8 mL of DPPH solution was added. After mixing well, the mixture was placed in the dark at room temperature for 90 minutes. The absorbance was measured at 517 nm and recorded as A1. The blank control was not added to the test solution and recorded as A0. Clearance rate calculation formula: Clearance rate (%) = 100% Diabetic foot was diagnosed according to the World Health Organization criteria. Ninety patients with diabetic foot were selected, with a disease course of 5-30 days, an age of 38-55 years, 55 males and 35 females. They were randomly divided into 9 groups with 10 patients in each group. There were no statistically significant differences in the general characteristics of the patients in each group, and they were comparable. Exclusion criteria for cases: ① Patients with grade 5 diabetic foot ulcers requiring amputation due to severe lesions; ② Patients and their families who refuse to cooperate with treatment and abandon treatment; ③ Patients with serious diseases such as heart, brain, kidney, hematopoietic system and endocrine system diseases; ④ Pregnant or lactating women; ⑤ Patients with a history of mental illness or a family history of mental illness; ⑥ Patients with cancerous, tuberculous and other specific ulcers; ⑦ Patients with allergic constitutions.

[0031] The Wagner classification system is used for grading diabetic foot. Grade 0: Risk factors for ulceration are present; Grade 1: Ulcers on the surface of the skin on the foot, but without signs of infection; Grade 2: Characterized by deep penetrating ulcers, often accompanied by soft tissue infection, but without osteomyelitis or deep abscess; Grade 3: Deep ulcers often affect bone tissue and may involve deep abscesses or osteomyelitis; Grade 4: Characterized by ischemic peptic ulcer gangrene, often accompanied by neuropathy without severe pain, and infection may be present on the surface of the necrotic tissue; Grade 5: Gangrene affects the entire foot; the lesions are extensive and severe, and some develop rapidly.

[0032] Nursing care: Dietary Care: Provide patients with strict dietary management and treatment. The daily calorie requirement for diabetic foot patients can be met by increasing the calorie intake by 10%-20% based on the patient's ideal weight. Combine this with their usual eating habits and activity levels to rationally plan daily meals, ensuring a diverse diet, reducing high-fat foods, abstaining from smoking and alcohol, drinking plenty of water, maintaining regular and measured meals, and ensuring a balanced nutritional intake to improve physical fitness, enhance disease resistance, and control blood sugar. Foot Care: Instruct patients and their families to examine their feet, avoid burns, pay attention to hygiene, and keep feet warm. Strict Blood Sugar Control: Fasting blood glucose should be controlled below 7 mmol / L, and postprandial 2-hour blood glucose should be controlled below 10 mmol / L, avoiding excessive fluctuations.

[0033] treat: Groups 1-8 were treated with the wound dressings prepared in Examples 1-4 and Comparative Examples 1-4, respectively. After cleaning the wound, necrotic tissue was removed from the surface. The wound was then cleaned with hydrogen peroxide solution and 0.9% sodium chloride solution in sequence. The wound was covered with medical dressing and fixed. When there was a lot of secretion, the dressing was changed once a day. When there was little secretion, the dressing was changed every 2 days.

[0034] Group 9 was designated as the control group: After cleaning the wound, necrotic tissue on the surface was removed, and the wound was cleaned sequentially with hydrogen peroxide solution and 0.9% sodium chloride solution. Regular insulin was applied locally as a wet dressing, and the wound was wrapped with sterile gauze and secured with tape.

[0035] Criteria for evaluating therapeutic efficacy: Significant effect: Symptoms disappear or become less noticeable, and wound healing rate is 70%-100%; Effective: Symptoms are significantly reduced, and wound healing rate is 30%-70%; Ineffective: The treatment was ineffective, did not meet the effective criteria, or the condition worsened and the infection was not effectively controlled.

[0036] Effectiveness = (Significantly Effective + Effective) / Total Number × 100% Table 1. Summary of experimental data from Examples 1-4 and Comparative Examples 1-4

[0037] Table 2 Summary of experimental data from Examples 1-4 and Comparative Examples 1-4

[0038] Data Analysis: As can be seen from Tables 1 and 2, the wound dressing prepared by this invention has the ability to heal wounds faster, higher permeability and absorbability, and stronger anti-inflammatory and antibacterial properties. This may be because the hydrogel in the first contact layer is prepared from methacrylamide gelatin and oxidized Angelica sinensis polysaccharide. Among them, Angelica sinensis polysaccharide can effectively promote cell proliferation, regulate immune response, promote angiogenesis and has antioxidant effects. Moreover, the hydrogel can simulate the extracellular matrix structure and has moisturizing, permeable, soft, absorbable and good biocompatibility. Hirudin has multiple effects such as wound repair, anti-fibrosis, and antithrombosis, and can also inhibit various diabetes-related complications. However, it has a short half-life in vivo, is easily degraded in the gastrointestinal tract, has poor mucosal permeability and low bioavailability. The modified hirudin liposomes effectively improve its drug stability and bioavailability, and work synergistically with the hydrogel. This product promotes the recovery of both fast and slow wounds. The intermediate transition layer, with its asymmetrical pore size fiber membrane structure, guides exudate from the first contact layer to the backing layer without backflow. In the backing layer, polyacrylamide is biodegradable and biocompatible, sodium alginate promotes wound healing, moisturizes, and absorbs moisture, while Bifidobacterium bifidum BD-1 inhibits common pathogens causing wound infections and promotes wound healing by regulating the inflammatory response and limiting pathogen colonization. The backing layer, prepared from polyacrylamide, sodium alginate, and Bifidobacterium bifidum, absorbs a large amount of exudate, providing a moist environment for the wound, inhibiting bacterial growth, and promoting wound healing. Through its unique three-layer structure design, combined with the synergistic effects of probiotics and various natural active ingredients, this wound dressing offers rapid wound healing, strong anti-inflammatory and antibacterial properties, high exudate absorption, and effective regulation of the wound microenvironment.

[0039] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0040] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A wound dressing for chronic wounds, characterized in that, The wound dressing is prepared from a first contact layer, an intermediate transition layer and a backing layer that are in direct contact with the wound. The first contact layer was prepared from methacrylamide gelatin, oxidized angelica polysaccharide and hirudin liposomes; The intermediate transition layer is a fiber membrane with asymmetrical pore sizes on both sides, with the side with larger pore size attached to the first contact layer and the side with smaller pore size attached to the backing layer. The backing layer is prepared from polyvinyl alcohol, sodium alginate and Bifidobacterium bifidum.

2. The wound dressing for chronic wounds according to claim 1, characterized in that, The method for preparing the hirudin liposomes is as follows: Step A1. Add lecithin and cholesterol to chloroform, disperse evenly by ultrasonication, and then use a rotary evaporator at 40-42℃ to remove the organic solvent until a uniform film is formed in the eggplant flask. Step A2. Add hirudin to a phosphate buffer solution with a pH of 7.2-7.4, and disperse it evenly by ultrasonication to obtain mixture A. Add mixture A along the wall of the bottle into a flask and hydrate it at 35-38℃ and 200-260 rpm for 30-40 minutes. After hydration, perform ultrasonication in an ice bath using an ultrasonic cell disruptor to obtain hirudin liposomes.

3. The wound dressing for chronic wounds according to claim 2, characterized in that, The ratio of lecithin, cholesterol, and chloroform used in step A1 is 100-150mg:60-90mg:50-90mL; The concentration of the phosphate buffer solution mentioned in step A2 is 0.02 mmol / L; The ratio of phosphate buffer solution to hirudin is 10-20 mL: 1-2 mg.

4. The wound dressing for chronic wounds according to claim 1, characterized in that, The method for preparing the first contact layer is as follows: Step B1. Add gelatin to a 0.02 mmol / L phosphate buffer solution with a pH of 7.2-7.4, and add methylpropyl anhydride dropwise while stirring. Stir at 600-800 rpm for 2-4 hours. After the reaction is complete, dialyze the reaction solution and freeze dry to obtain methacrylated gelatin. Step B2. Add sodium periodate to a 2% aqueous solution of Angelica polysaccharide and stir in the dark at 37-42℃ for 10-12 hours. After stirring, add ethylene glycol to stop the reaction, then add 95% ethanol and let stand at 0-4℃ for 18-20 hours. Centrifuge and freeze dry to obtain oxidized Angelica polysaccharide. Step B3. Add methacrylated gelatin to a phosphate buffer solution, sonicate for 3-5 minutes, then add oxidized angelica polysaccharide, hirudin liposomes and phenyl-2,4,6-trimethylbenzoylphosphonic acid, mix well to obtain the first contact layer.

5. The wound dressing for chronic wounds according to claim 4, characterized in that, The ratio of gelatin, phosphate buffer solution and methylpropionic anhydride used in step B1 is 1-1.5g:10-20mL:0.7-1.2mL; The ratio of sodium periodate, Angelica polysaccharide aqueous solution, ethylene glycol and ethanol used in step B2 is 0.6-1g:50-100mL:75-150mL:500-2000mL; The mass ratio of methacrylated gelatin, phosphate buffer solution, oxidized Angelica polysaccharide, hirudin liposomes and phenyl-2,4,6-trimethylbenzoylphosphonic acid in step B3 is 10-18:100-200:3-6:3-5.5:0.1-0.

15.

6. The wound dressing for chronic wounds according to claim 1, characterized in that, The intermediate transition layer is prepared as follows: Step C1. Add chitosan to a 50% acetic acid aqueous solution and stir at room temperature for 12-18 hours to obtain mixture B; then add polyethylene oxide to a 50% acetic acid aqueous solution and stir until completely dissolved to obtain mixture C. Add volumes V1 and V2 of mixture C to 10 mL of mixture B and stir for 3-5 hours. After standing for 18-24 hours, centrifuge to obtain spinning solution A and spinning solution B, respectively. Step C2. Using the dual-channel electrospinning method, add spinning solution A and spinning solution B to two 10mL syringes respectively, connect the electrospinning machine, set the electrospinning parameters, and perform electrospinning to obtain the intermediate transition layer.

7. The wound dressing for chronic wounds according to claim 6, characterized in that, The ratio of chitosan to acetic acid aqueous solution in step C1 is 3-5g:100mL; The ratio of the amount of polyethylene oxide to the aqueous acetic acid solution is 2-3 g: 100 mL; The value of V1 is 6.5-7.9 mL; V2 is 10 mL; In step C2, the electrospinning parameters are: voltage 21kV, spinning solution feed rate 1.5mL / h, distance from receiving roller to needle tip 15cm, and spinning time 2-3h.

8. The wound dressing for chronic wounds according to claim 1, characterized in that, The backing layer is prepared as follows: A 10% (w / w) aqueous solution of polyvinyl alcohol and a 2% (w / w) aqueous solution of sodium alginate were mixed and stirred at 37-42℃ for 2-3 hours. After cooling to room temperature, a suspension of Bifidobacterium bifidum was added, mixed evenly, and allowed to stand for 2-3 hours to remove air bubbles. The mixture was then poured into a mold, freeze-dried, and thawed at room temperature for 4 hours. This process was repeated for 3 cycles and freeze-dried again to obtain the backing layer.

9. The wound dressing for chronic wounds according to claim 8, characterized in that, The volume ratio of the polyvinyl alcohol aqueous solution, sodium alginate aqueous solution, and Bifidobacterium bifidum suspension is 20-30:20-35:3-5; The freeze-drying process is carried out at a temperature of -20 to 17°C for 18 to 20 hours.

10. The method for preparing a wound dressing for chronic wounds according to any one of claims 1-9, characterized in that, Includes the following steps: The first contact layer is attached to the side of the intermediate transition layer with larger pore size and photocured by irradiation with ultraviolet light at a wavelength of 365nm. Then, the backing layer is attached to the side of the intermediate transition layer with smaller pore size to obtain the wound dressing for chronic wounds.

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