A composite wound dressing and a method of making the same

Abstract

The application provides a composite wound dressing and a preparation method thereof, and belongs to the technical field of medical dressings. The composite wound dressing comprises an outer support layer and an inner functional layer. The outer support layer is a modified ramie fiber non-woven fabric, and the modification treatment comprises low-temperature plasma treatment and acetylation modification on the ramie fiber. The inner functional layer is a composite of ramie knitted fabric and low antigen collagen sponge grafted with carvacol. The collagen sponge is inside the yarn gap and pore of the knitted fabric, forming a structure in which the collagen sponge and the ramie fiber are wrapped and inserted with each other. The double-layer structure design of the outer support layer and the inner functional layer of the dressing can provide stable mechanical support through the outer layer, maintain the shape retention of the dressing, realize efficient liquid absorption through the inner layer, release the antibacterial components, actively promote wound healing, has excellent biocompatibility and low immunogenicity risk.

Description

Technical Field

[0001] This application relates to the field of medical dressing technology, and in particular to a composite wound dressing and its preparation method. Background Technology

[0002] Ideal wound dressings should mimic skin function and possess good biocompatibility, mechanical support, absorbency and breathability, antibacterial properties, and active healing capabilities. Collagen-based dressings and cellulose-based dressings are currently the mainstream products in biological dressings. Collagen is an excellent healing material due to its similarity to the extracellular matrix, but single collagen dressings have the following inherent defects: (1) poor mechanical strength, easy to dry and crack, and poor shape retention; (2) insufficient antibacterial properties, which can easily lead to infection; (3) weak ability to absorb tissue exudate; (4) fast degradation rate and poor stability; (5) animal-derived collagen has the risk of immunogenicity. Cellulose-based dressings (such as bacterial cellulose dressings) have good antibacterial and hygroscopic properties, but they have disadvantages such as poor skin affinity and bioactivity compared to collagen, certain sources (such as bacterial cellulose) have certain immunogenicity, and they lack biological signals that actively promote cell proliferation and wound healing.

[0003] Ramie fiber possesses high strength, high moisture absorption, good breathability, and natural antibacterial properties (containing benzoyl, pyrimidine, purine, and other components), making it an ideal reinforcing material for dressings. However, ramie fiber has high crystallinity, high stiffness, and numerous surface hairs, leading to intense itching sensations in fabrics. Direct contact with wounds can cause mechanical irritation and secondary damage. Existing methods for reducing itching (alkali treatment, sand washing, chemical reagent treatment) suffer from significant fiber damage, severe pollution, or high costs. Publicly available patents (such as ZL202211372027.4 and CN119102118A) employ a composite bio-enzyme treatment method, which is effective but involves complex procedures and long processing cycles.

[0004] The leather industry generates a large amount of limed leather scraps, rich in collagen, which are currently mostly discarded or processed at low value (such as into industrial gelatin), resulting in resource waste and environmental pollution. Finding ways to utilize this waste in a high-value, resource-efficient manner, and combining it with locally abundant ramie resources to develop high-performance medical products, has significant economic and social value.

[0005] Existing collagen dressings mostly use physical mixing of nano-silver, antibiotics and other antibacterial agents, which have problems such as sudden release of antibacterial agents, safety risks (nano-silver cytotoxicity) and short antibacterial duration. Summary of the Invention

[0006] This application is made in view of the above-mentioned problems, and its purpose is to provide a composite wound dressing and a method for preparing the same.

[0007] Specifically, the first aspect of this application provides a composite wound dressing, including an outer support layer and an inner functional layer; The outer support layer is a modified ramie fiber nonwoven fabric, and the modification treatment includes low-temperature plasma treatment and acetylation modification of the ramie fibers; The inner functional layer is a composite of ramie woven fabric and low-antigen collagen sponge grafted with carvacrol. The collagen sponge penetrates through the gaps and pores of the yarn in the woven fabric, forming a structure in which the collagen sponge and ramie fibers wrap around and interweave with each other.

[0008] Furthermore, the low-antigen collagen contains residual calcium ions; The carvacrol is covalently grafted onto the carboxyl group of the collagen via an esterification reaction; The calcium ions and carvacrol form a synergistic antibacterial effect in the slightly acidic environment of the wound.

[0009] Furthermore, the low-temperature plasma treatment is carried out in an oxygen or nitrogen atmosphere, with a treatment power of 100~300W and a treatment time of 2~10min.

[0010] Furthermore, the raw material for extracting the low-antigen collagen is scraps of leather from tanning and liming processes.

[0011] Furthermore, the inner functional layer undergoes a secondary cross-linking treatment with a cross-linking agent, the amount of which is 0.10% to 0.35% of the dry weight of the collagen.

[0012] A second aspect of this application provides a method for preparing the composite wound dressing, comprising the following steps: S1: Ramie fibers are subjected to enzymatic degumming treatment, steam explosion treatment, low-temperature plasma treatment and acetylation modification in sequence to obtain modified ramie fibers. The modified ramie fibers are formed as an outer support layer. At the same time, a portion of the modified ramie fibers are woven into a woven fabric. S2: Using leather scraps from tanning and liming as raw materials, the residual calcium ion content in the raw materials is controlled at 0.8%~1.2% of the dry weight through deliming treatment. Then, the raw materials are extracted, purified and dialyzed by alkaline protease to obtain a low antigen collagen solution. S3: In the presence of a crosslinking agent, carvacrol is covalently grafted onto the carboxyl group of the low-antigen collagen to obtain a carvacrol-grafted collagen solution. S4: The collagen solution grafted with carvacrol obtained in step S3 is filled into the woven fabric obtained in step S1. After vacuum degassing, it is freeze-dried using a freeze-drying process. After drying, a composite of collagen sponge and ramie three-dimensional skeleton interpenetrates, which serves as the inner functional layer. S5: Combine the inner functional layer obtained in step S4 with the outer support layer obtained in step S1, and sterilize to obtain the composite wound dressing.

[0013] Further, the deashing treatment in step S2 includes: first treating the scraps of the ash-soaked skin with a 1.0%~2.5% ammonium sulfate solution for 10~30 min, then treating them with a buffer solution containing 0.3%~0.8% sodium citrate and 0.05~0.15 mol / L calcium chloride for 20~60 min, monitoring the endpoint with a calcium ion selective electrode or EDTA titration method, so that the residual calcium ion content in the raw material is controlled at 0.8%~1.2% of the dry weight.

[0014] Furthermore, the parameters for alkaline protease hydrolysis extraction are as follows: enzyme addition amount is 8%~15% of the dry weight of the raw material, temperature is 33~40℃, pH is 8.5~9.5, and time is 4~6 hours.

[0015] Furthermore, the freeze-drying process in step S4 includes: Pre-freezing stage: Pre-freeze at -20~-30℃ for 2~4 hours; Deep freezing stage: Cool to -40~-60℃ at a rate of 2~5℃ / h and deep freeze for 4~8 hours; Gradient heating: Gradually increase the temperature to 20℃~25℃ at a heating rate of 1~3℃ / h, with a vacuum degree ≤20 Pa.

[0016] Furthermore, in step S1, the acetylation modification uses acetic anhydride as the acetylation agent and reacts at 50-70°C for 30-90 min in the presence of a catalyst.

[0017] Furthermore, in step S3, the crosslinking agent used for the carvacrol grafting reaction is EDC / NHS, the reaction temperature is 25~37℃, and the reaction time is 4~12 hours.

[0018] Furthermore, in step S3, the collagen solution grafted with carvacrol undergoes a secondary cross-linking treatment with a cross-linking agent before step S4. The cross-linking agent used in the secondary cross-linking treatment is genipin, with an addition amount of 0.10%~0.35% of the dry weight of collagen, a reaction temperature of 20~30℃, and a reaction time of 4~8 hours.

[0019] The present invention has the following beneficial effects: This invention utilizes low-antigen collagen extracted from leather scraps after tanning and liming, achieving high-value resource utilization of tanning waste. It also combines the natural high strength, high moisture absorption, and breathability of ramie to form a composite structure. This solves the problems of insufficient mechanical strength and rapid degradation in single-collagen dressings, while overcoming the shortcomings of insufficient bioactivity and poor skin affinity in pure ramie dressings. Carvacrol is covalently grafted onto the collagen, avoiding the problem of sudden release of antibacterial agents. Simultaneously, the calcium ions remaining in the collagen and carvacrol form a synergistic antibacterial effect in the slightly acidic environment of the wound, resulting in a longer-lasting antibacterial effect and higher safety. Furthermore, the ramie fibers are sequentially enzymatically degraded... The process involves gluing, steam explosion, low-temperature plasma treatment, and acetylation modification. While introducing polar groups to improve compatibility, it also allows for controllable regulation of ramie degradation rates, significantly reducing the crystallinity and stiffness of ramie fibers, improving irritation, and simplifying the process. The process is simple and clean, avoiding excessive fiber damage and environmental pollution. The dual-layer structure design, consisting of an outer support layer and an inner functional layer, provides stable mechanical support through the outer layer to maintain the dressing's shape, while the inner layer enables efficient absorption of exudate and slow-release of antibacterial components to actively promote wound healing. It exhibits excellent biocompatibility, low immunogenicity risk, and promising application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this drawing or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this drawing. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0021] Figure 1 Infrared spectra before and after collagen grafting modification; Figure 2 Image of the inner layer of collagen sponge; Figure 3 This is an image of the inner layer of the electrospun yarn.

[0022] The purpose, features, and advantages of this accompanying drawing will be further explained in conjunction with the embodiments and with reference to the accompanying drawing. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the following description and illustration are provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0024] Obviously, the following description is merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios without any inventive effort. Furthermore, it is understood that although the effort involved in such development may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0025] An embodiment of the first aspect of this application provides a composite wound dressing, comprising an outer support layer and an inner functional layer; The outer support layer is a modified ramie fiber nonwoven fabric, and the modification treatment includes low-temperature plasma treatment and acetylation modification of the ramie fibers; The inner functional layer is a composite of ramie woven fabric and low-antigen collagen sponge grafted with carvacrol. The collagen sponge penetrates through the gaps and pores of the yarn in the woven fabric, forming a structure in which the collagen sponge and ramie fibers wrap around and interweave with each other.

[0026] The composite wound dressing provided by this invention provides stable mechanical support through an outer support layer, effectively maintaining the overall shape of the dressing and preventing deformation and damage due to swelling from absorbing liquid during use. It conforms to the wound surface while meeting the needs of dressing changes. The inner layer's structure, utilizing the high hygroscopicity of the ramie skeleton, rapidly absorbs tissue exudate from the wound, maintaining a dry and clean healing environment. Simultaneously, carvacrol grafted onto collagen is slowly and continuously released, combining with the synergistic antibacterial effect of residual calcium ions in the collagen to persistently inhibit bacterial proliferation in the wound, while avoiding the safety issues associated with sudden release of antibacterial agents. This dual-layer structure works synergistically, possessing mechanical stability, high hygroscopicity, long-lasting antibacterial properties, and active healing-promoting capabilities, making it suitable for the healing needs of different types of wounds.

[0027] In some embodiments, the low-antigen collagen contains residual calcium ions; carvacrol is covalently grafted onto the carboxyl group of the collagen via esterification; the calcium ions and carvacrol form a synergistic antibacterial effect in the slightly acidic environment of the wound. The low-antigen collagen retains an appropriate amount of calcium ions from the raw material used in leather tanning and pyrolysis, achieving a synergistic effect with carvacrol without additional addition. When the wound becomes infected and presents a slightly acidic environment, calcium ions are gradually released, disrupting the permeability of the bacterial cell membrane and assisting carvacrol in entering the bacterial cell to exert its antibacterial effect, significantly enhancing the antibacterial efficacy.

[0028] In some embodiments, the acetylation modification of ramie fibers can change the crystallinity of ramie fibers by adjusting the degree of acetylation substitution, thereby adjusting the degradation rate of ramie in vivo to match the wound healing cycle, avoiding premature degradation and loss of support or slow degradation affecting the growth of new tissue, eliminating the need for secondary dressing changes to remove residual dressings, and reducing secondary stimulation to the wound.

[0029] In some embodiments, the low-temperature plasma treatment of the ramie fibers is carried out in an oxygen or nitrogen atmosphere, with a treatment power of 100-300W and a treatment time of 2-10 minutes. This treatment can introduce a large number of polar groups such as hydroxyl and amino groups onto the fiber surface without damaging the mechanical properties of the ramie fibers, thereby enhancing the interfacial bonding force between the ramie fibers and collagen, preventing the collagen phase and ramie phase from delaminating and falling off during use, and ensuring the stability of the dressing structure.

[0030] In some embodiments, the raw material for extracting the low-antigen collagen is scraps from tannery leather. Collagen is obtained by extracting and purifying tannery waste, preserving its good biological activity while eliminating the complex process of separately removing immunogens from animal-derived collagen. This reduces production costs and avoids resource waste and environmental pollution caused by large quantities of scraps from tannery leather, achieving high-value resource utilization of waste.

[0031] In some embodiments, the inner functional layer undergoes a secondary cross-linking treatment with a cross-linking agent, wherein the amount of the cross-linking agent added is 0.10% to 0.35% of the dry weight of the collagen. This secondary cross-linking treatment can further enhance the structural stability of the collagen sponge, regulate its degradation rate, prevent rapid degradation and disintegration of collagen in wound exudate, and simultaneously ensure that the grafted carvacrol is continuously released along with the slow degradation of collagen, thus prolonging the duration of antibacterial action and meeting the needs of long-term wound care.

[0032] An embodiment of the second aspect of this application provides a method for preparing the composite wound dressing, comprising the following steps: S1: Ramie fibers are subjected to enzymatic degumming treatment, steam explosion treatment, low-temperature plasma treatment and acetylation modification in sequence to obtain modified ramie fibers. The modified ramie fibers are formed as an outer support layer. At the same time, a portion of the modified ramie fibers are woven into a woven fabric. S2: Using leather scraps from tanning and liming as raw materials, the residual calcium ion content in the raw materials is controlled at 0.8%~1.2% of the dry weight through deliming treatment. Then, the raw materials are extracted, purified and dialyzed by alkaline protease to obtain a low antigen collagen solution. S3: In the presence of a crosslinking agent, carvacrol is covalently grafted onto the carboxyl group of the low-antigen collagen to obtain a carvacrol-grafted collagen solution. S4: The collagen solution grafted with carvacrol obtained in step S3 is filled into the woven fabric obtained in step S1. After vacuum degassing, it is freeze-dried using a freeze-drying process. After drying, a composite of collagen sponge and ramie three-dimensional skeleton interpenetrates, which serves as the inner functional layer. S5: Combine the inner functional layer obtained in step S4 with the outer support layer obtained in step S1, and sterilize to obtain the composite wound dressing.

[0033] In this embodiment, in step S1: Degumming: Ramie fibers are subjected to routine degumming treatment to remove most of the pectin and hemicellulose.

[0034] Enzymatic hydrolysis and softening: Degummed ramie fibers were immersed in a treatment solution containing pectinase (2 g / L) and nonionic surfactant JFC (2 g / L) at a bath ratio of 1:20. The pH was adjusted to 4.0 with 0.1 mol / L Britton-Robinson buffer, and the solution was kept at a constant temperature of 50°C with a stirring speed of 500 r / min for 60 min. During the treatment, pectinase hydrolyzed the gum complex on the fiber surface, releasing the fuzz.

[0035] Steam explosion physical modification: After enzyme treatment, the fibers are washed with water and placed in a steam explosion device. The pressure is controlled at 1.8~2.2 MPa, and the pressure is maintained for 30 seconds before being released instantly. During the explosion process, the free water inside the fiber rapidly vaporizes, impacting the fiber surface, further removing residual fuzz and increasing the surface roughness of the fiber.

[0036] Low-temperature plasma surface etching: The fiber after steam explosion is placed in a plasma treatment chamber, oxygen or nitrogen is introduced, power is 100~300 W, treatment time is 2~10 min, polar groups (-OH, -COOH) are introduced on the fiber surface and micro-pit structure is formed.

[0037] Acetylation modification: Plasma-treated ramie fibers were immersed in anhydrous toluene solution containing 5% (v / v) acetic anhydride and 0.1% (v / v) pyridine (catalyst) at a bath ratio of 1:15 and reacted at 60°C for 45 minutes. After the reaction, the fibers were washed three times with anhydrous ethanol, rinsed with deionized water, and dried at 50°C. The acetyl content was determined by acid-base titration, with the DS controlled between 0.15 and 0.25. At this degree of substitution, the degradation half-life of ramie fibers in cellulase solution (1 U / mL, 37°C) was shortened from 35 days before modification to 21 days, matching the degradation half-life of inner collagen (14.6 days).

[0038] Modified fiber web formation: Modified ramie fibers are opened, carded, and air-laid (weight controlled at 30~80 g / m²), then reinforced by hydroentangling (pressure 50~150 bar, 3~5 passes), and dried to obtain ramie nonwoven outer layer substrate.

[0039] Table 1 shows a comparison of the effects of ramie fiber before and after treatment in step S1.

[0040]

[0041] In this embodiment, in step S2: Raw materials: scraps of cowhide after tanning and liming (taken from a local tannery, already dehaired and limed) cut into small pieces of about 3cm x 3cm.

[0042] The deliming process includes: first, treating the shredded, limed hide scraps with a 1.0%–2.5% ammonium sulfate solution for 10–30 minutes to remove free alkali from the surface; then treating them with a buffer solution (pH 7.0) containing 0.3%–0.8% sodium citrate and 0.05–0.15 mol / L calcium chloride for 20–60 minutes, utilizing the competitive complexation and replacement of some structural calcium by citrate ions, while calcium chloride maintains the calcium ion activity in the solution, preventing excessive leaching of bound calcium. The endpoint is monitored using a calcium ion selective electrode or EDTA titration method to control the residual calcium ion content in the raw material at 0.8%–1.2% of dry weight. The raw material is then rapidly rinsed twice with deionized water (5 minutes each time), dried, and pulverized through a 40-mesh sieve to obtain partially decalcified hide powder.

[0043] Enzymatic extraction: Decalcified bovine hide powder was suspended in 0.1 mol / L Briton-Robinsin buffer (pH 9.0) at a material-to-liquid ratio of 1:12, with an additional 0.5 mmol / L CaCl2 added to maintain stable calcium coordination during extraction. Alkaline protease (enzyme activity ≥ 200,000 U / g) was added at 10% (w / w) of the dry weight of the hide powder. The reaction was carried out at 33–40 °C and a stirring speed of 440 r / min for 4–6 hours.

[0044] Inactivation and purification: After the reaction, the temperature was raised to 85℃ and held for 10 min for inactivation. The mixture was filtered through four layers of gauze, and the filtrate was centrifuged at 4℃ and 10,000 rpm for 15 min. Sodium chloride was slowly added to the supernatant to a final concentration of 0.8 M, and the mixture was allowed to stand overnight to allow collagen to salt out and precipitate. The mixture was then centrifuged again (4℃, 10,000 rpm, 15 min), and the precipitate was collected.

[0045] Dialysis: Dissolve the precipitate in 0.5 mol / L acetic acid, place it in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyze against deionized water at 4℃ for 48-72 hours (changing the water every 8 hours). After dialysis, adjust the solids concentration of the collagen solution to 3%-6%.

[0046] In this embodiment, in step S3: Grafting reaction: Dilute the above low-antigen collagen solution to 2 mg / mL with PBS buffer (pH 6.5). Add cross-linking agent EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) (final concentration 0.5 mg / mL) and carvacrol (final concentration 0.5~2 mg / mL, pre-dissolved in a small amount of ethanol), and stir at 25~37℃ for 6~12 hours.

[0047] Purification: The reaction solution was dialyzed with deionized water (MWCO 3500 Da) for 24 hours to remove unreacted carvacrol and small molecule cross-linking agents. The mixture was then lyophilized to obtain the carvacrol-grafted modified collagen sponge precursor.

[0048] See Figure 1 FT-IR spectra are in the range of 1730–1750 cm⁻¹ -1 The appearance of a new ester bond absorption peak confirms that the phenolic hydroxyl group of carvacrol has undergone esterification and covalent bonding with the carboxyl group of collagen. Simultaneously, the phenolic hydroxyl peak (3500-3200 cm⁻¹) is observed. -1 The intensity weakened, further confirming that grafting had occurred.

[0049] Genipin secondary crosslinking: The grafted collagen sponge precursor was redissolved in PBS (pH 7.4) to a concentration of 3% (w / v), and genipin was added at an amount of 0.15%~0.25% (w / w) of the collagen dry weight. The reaction was stirred at 25°C for 6 hours. After the reaction, the mixture was dialyzed again (MWCO 3500 Da, 4°C, 12 hours) to remove free genipin, and then lyophilized.

[0050] The free amino content was determined by the trinitrobenzenesulfonic acid method, and the degree of crosslinking was controlled at 18%~22%. Under this degree of crosslinking, the degradation half-life of collagen sponge in PBS containing type I collagenase (37℃, 0.5 U / mL) was extended from 4.2 days before modification (uncrosslinked) to 14.6 days.

[0051] In this embodiment, in step S4: Ramie fiber fabric weaving: The modified ramie fibers from step S1 are spun into 20-40 Nm yarns and woven into three-dimensional spaced fabrics on a warp knitting machine. Parameters: areal density 200-400 g / m². 2 Thickness 3~8 mm, warp and weft density 40~60×30~50 threads / 10cm.

[0052] Collagen impregnation and filling: A collagen solution grafted with carvacrol (concentration 3%~6%) is evenly poured into the three-dimensional woven fabric, followed by vacuum degassing to ensure the collagen solution fully fills the fabric pores. See also Figure 2 , Figure 2 This is an image of the inner layer of a collagen sponge.

[0053] Gradient temperature-controlled freeze-drying: Pre-freezing: -20~-30℃, 2~4 hours; Deep freezing: cooling to -40~-60℃ at a rate of 2~5℃ / h for 4~8 hours; Gradient heating: gradually heating to 20℃~25℃ at a rate of 1~3℃ / h, with a vacuum degree ≤20 Pa. After freeze-drying, a composite skeletal structure of interpenetrating collagen sponge and fabric is formed.

[0054] In this embodiment, step S5, composite outer layer: cover one side of the composite skeleton with the outer support layer obtained in step S1, spray a small amount of medical grade polyacrylate adhesive (or genipin solution) between the composite skeleton and the outer support layer, and hot press composite at 40~60℃ and 0.5~2 MPa for 5~10 min.

[0055] Sterilization: Irradiation with cobalt-60 gamma rays, dose 10~25 kGy.

[0056] In another preferred embodiment, steps S4 and S5 can be: Inner layer preparation: The collagen solution grafted with carvacrol was dissolved in hexafluoroisopropanol (HFIP) to prepare an 8-12 wt% spinning solution. Electrospinning equipment was used with a spinning voltage of 13-25 kV, a receiving distance of 15-20 cm, a solution supply rate of 0.5-1.5 mL / h, a roller speed of 100-200 r / min, and a spinning time of 2-4 h. The electrospun membrane was collected on aluminum foil. The fiber diameter was controlled at 300-800 nm, and the membrane thickness was approximately 0.1-0.3 mm. See also... Figure 3 , Figure 3 This is an image of the inner layer of an electrospun film.

[0057] Composite: A small amount of medical-grade polyacrylate adhesive (or genipin solution) is sprayed between the electrospun film and the ramie fiber woven fabric, and then hot-pressed at 40~60℃ and 0.5~2 MPa for 5~10 min.

[0058] Sterilization: Irradiation with cobalt-60 gamma rays, dose 10~25 kGy.

[0059] Example The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight. Unless otherwise stated, all reagents used in the examples are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, all instruments used in the examples are available commercially.

[0060] The following examples and comparative examples contain some of the raw materials sourced from: Grey cowhide scraps: sourced from a tannery in Zhejiang Province, already dehaired and limed, with an initial calcium content of approximately 3.2% (dry weight). Ramie fiber: provided by a ramie company in Hunan; Alkaline protease (enzyme activity ≥ 200,000 U / g): Novozymes; Carvacrol (purity ≥98%): Sigma-Aldrich; EDC, NHS, Genipin: Shanghai Aladdin Reagents; Medical grade polyacrylate adhesive: LOCTITE AA3321.

[0061] Example 1 A method for preparing the composite wound dressing includes the following steps: S1: Degummed ramie fibers were immersed in a treatment solution containing pectinase (2 g / L) and nonionic surfactant JFC (2 g / L) at a bath ratio of 1:20. The pH was adjusted to 4.0 with 0.1 mol / L Britton-Robinson buffer. The solution was kept at a constant temperature of 50°C in a water bath, with a stirring speed of 500 r / min for 60 min. After enzyme treatment, the fibers were washed with water and placed in a steam explosion device. The pressure was controlled at 1.8 MPa and held for 30 s before being released instantaneously. The fibers after steam explosion were placed in a plasma treatment chamber at a power of 200 W for 5 min. The plasma-treated ramie fibers were then immersed in an anhydrous toluene solution containing 5% (v / v) acetic anhydride and 0.1% (v / v) pyridine at a bath ratio of 1:15 and reacted at 60°C for 45 min. The acetyl content was determined by acid-base titration, and the DS was controlled to be 0.21 to obtain modified ramie fibers. Modified ramie fibers are opened, carded, and air-laid into a web, then reinforced by hydroentangling (100 bar pressure, 4 passes), and dried to obtain a ramie nonwoven outer layer substrate; a portion of the fibers is spun into yarn (30 Nm) and warp-knitted into a three-dimensional spacer fabric (area density 300 g / m²). 2 (5mm thick, warp and weft density 50×40 threads / 10cm), woven into a woven fabric; S2: The scraps of slaked hide were treated with a 1.5% ammonium sulfate solution at a bath ratio of 1:10 for 20 min; then treated with a buffer solution containing 0.5% sodium citrate and 0.1 mol / L calcium chloride (pH 7.0) for 40 min, monitored by a calcium ion electrode. The endpoint calcium residue was 1.05%. The hide was then rapidly rinsed twice with deionized water (5 minutes each time), dried, and pulverized through a 40-mesh sieve to obtain partially decalcified hide powder. The decalcified hide powder was suspended in 0.1 mol / L Briton-Robinsin buffer (pH 9.0) and 0.5 mmol / L CaCl2 at a material-to-liquid ratio of 1:12. Alkaline protease (enzyme activity ≥200,000 U / g) was added at 10% (w / w) of the dry weight of the hide powder. The reaction was carried out at 35℃ and a stirring speed of 440 r / min for 5 hours. After the reaction, the temperature was raised to 85℃ and held for 10 min to inactivate the enzyme. The solution was filtered through four layers of gauze, and the filtrate was centrifuged at 10,000 rpm for 15 min at 4 °C. Sodium chloride was slowly added to the supernatant to a final concentration of 0.8 M, and the solution was allowed to stand overnight to allow collagen to salt out and precipitate. The solution was centrifuged again (4 °C, 10,000 rpm, 15 min), and the precipitate was collected. The solution was dissolved in 0.5 mol / L acetic acid, placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed against deionized water at 4 °C for 72 hours (changing the water every 8 hours). After dialysis, the solids concentration of the collagen solution was adjusted to 4.5%, and the calcium content was measured to be 0.98% by ICP-OES, yielding a low-antigen collagen solution. S3: The low-antigen collagen solution was diluted to 2 mg / mL with PBS buffer (pH 6.5). Crosslinking agents EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) (final concentration 0.5 mg / mL) and carvacrol (final concentration 0.5–2 mg / mL, pre-dissolved in a small amount of ethanol) were added, and the mixture was stirred at 30°C for 8 hours. The reaction solution was dialyzed against deionized water (MWCO 3500 Da) for 24 hours; lyophilized, and redissolved in PBS (pH 7.4) to a concentration of 3% (w / v). Genipin 0.2% (w / w) was added, and the mixture was stirred at 25°C for 6 hours. The reaction solution was dialyzed again (MWCO 3500 Da, 4°C, 12 hours); lyophilized, and the free amino content was determined using the trinitrobenzenesulfonic acid method, with the crosslinking degree controlled at 20%. S4: The grafted crosslinked collagen solution from step S3 above was diluted to 4.5% with PBS and injected into the woven fabric obtained in step S1. Vacuum degassing was performed at 0.09 MPa for 15 min, followed by freeze-drying using the following process: Pre-freezing: -25℃ for 3 hours; Deep freezing: cooling to -40℃ at a rate of 5℃ / h for 8 hours; Gradient heating: gradually heating to 20℃ at a rate of 2℃ / h, with a vacuum degree ≤20 Pa, serving as the inner functional layer. S5: Spray the inner functional layer obtained in step S4 and the outer support layer obtained in step S1 with medical-grade polyacrylate adhesive, hot-press them together at 50°C and 1MPa for 8 minutes, and sterilize them by irradiation with 15kGy of cobalt-60 to obtain the composite wound dressing.

[0062] Example 2 This embodiment is basically the same as that of Embodiment 1, except that in step S2, during the deashing process, a buffer solution (pH 7.0) containing 0.5% sodium citrate and 0.1 mol / L calcium chloride was used for 70 min, monitored by a calcium ion electrode, and the final calcium residue was 0.52%; no CaCl2 was added to the subsequent enzymatic digestion buffer.

[0063] Example 3 This embodiment is basically the same as that of embodiment 1, except that in step S2, during the deashing process, the scraps of the ash-soaked skin are treated with 0.5% ammonium sulfate solution at a bath ratio of 1:10 for 10 minutes without being treated with sodium citrate buffer. The calcium ion electrode is used for monitoring, and the final calcium residue is 1.92%. The concentration of CaCl2 in the subsequent enzymatic hydrolysis buffer is increased to 1.0 mmol / L to maintain calcium stability.

[0064] Example 4 This embodiment is basically the same as that of embodiment 1, except that the acetylation reaction in step (1) is carried out at 60°C for 25 minutes (DS measured 0.13). Step (3) The amount of genipin added was reduced to 0.12%.

[0065] Comparative Example 1 This comparative example is basically the same as Example 1, except that the deashing treatment in step S2 is to treat the ash-soaked scraps with a 4% ammonium sulfate solution at a bath ratio of 1:10 for 60 minutes. Step S3 does not involve chemical grafting of carvacrol, but instead involves physical mixing: before freeze-drying the collagen solution, add an equal amount (1.0 mg / mL) of carvacrol, stir briefly to mix well, and then freeze-dry directly.

[0066] Comparative Example 2 This comparative example is basically the same as Example 1, except that after the carvacrol grafting in step S3, the secondary cross-linking of genipin is not performed (direct freeze-drying). In step S1, the ramie fibers are not acetylated (they are directly web-formed after plasma treatment).

[0067] Comparative Example 3 This comparative example is basically the same as Example 1, except that the fiber is spun but not woven into a three-dimensional warp-knitted spaced fabric, and the grafted crosslinked collagen solution in step S4 is diluted with PBS to 4.5% and then directly freeze-dried.

[0068] Comparative Example 4 This comparative example is basically the same as Example 1, except that in step S1, the outer ramie is only subjected to conventional degumming and opening to form a web, without steam explosion, plasma, or acetylation treatment (only conventional enzymatic hydrolysis with 2g / L pectinase for 30min is performed).

[0069] Experimental Case The following tests were conducted on the samples prepared in Examples 1-4 and Comparative Examples 1-4 above: (1) Compression strength determination: The freeze-dried sponge sample to be tested was cut into standard cylindrical specimens using a circular punch with a diameter of 20 mm. The height of the specimen was measured with vernier calipers to ensure it was within the range of 5.0 ± 0.2 mm. A universal testing machine (Instron 5967 model, equipped with a 50 kN sensor) was used for compression testing. A flat compression fixture with a diameter of 50 mm (the upper and lower pressure plates are stainless steel parallel plates) was selected, and the compression speed was set to 1.0 mm / min. The specimen was placed in the center of the lower pressure plate, and the upper pressure plate was lowered with a preload force of 0.1 N to contact the surface of the specimen to eliminate the unevenness error of the specimen surface. Then, it was compressed at a constant speed of 1.0 mm / min until the strain reached 50% (corresponding to a displacement of 2.5 mm). The testing machine automatically records the real-time load-displacement curve, reads the compressive load F (N) corresponding to the compression strain reaching 30% (i.e., displacement 1.5 mm), and calculates the compressive stress σ = F / A according to the formula. Five parallel samples are tested in each group of samples. After removing the maximum and minimum values, the arithmetic mean of the remaining three is taken as the final result.

[0070] (2) Degradation half-life determination: The degradation half-life was determined independently for the inner functional layer and the outer support layer of the dressing.

[0071] Degradation test of inner collagen sponge: The sample was cut into cubes of 1.0 cm × 1.0 cm × 0.5 cm, and its initial dry weight (m0, weighed after drying to constant weight in an oven at 50℃) was accurately measured. The sample was then immersed in phosphate buffer (PBS, pH 7.4, containing 0.02% sodium azide to inhibit the growth of other bacteria) containing 0.5 U / mL type I collagenase (derived from Clostridium histolyticum, Sigma C9891) at a bath ratio of 1:100 (i.e., 1 mL of enzyme solution was added for every 10 mg of sample) and placed in a constant temperature shaker at 37℃ at 100 r / min for degradation.

[0072] Degradation test of outer ramie fiber: The fiber was cut into 3.0 cm lengths, and the initial dry weight m0 was accurately weighed. It was then immersed in an acetate buffer (0.1 mol / L, pH 4.8) containing 1.0 U / mL cellulase (derived from Trichoderma viride, Sigma C2730) at a liquor ratio of 1:100 and degraded by shaking at 37℃ and 100 r / min.

[0073] For both types of degradation experiments, samples were taken every 24 hours. Each sample was rinsed three times with deionized water to remove adhering enzyme solution and degradation debris. After drying in a 50℃ oven to constant weight, the remaining mass m_t was weighed, and the mass retention rate R (%) was calculated as: R = m_t / m0 × 100%. A linear regression was performed on the natural logarithm of the retention rate ln(R / 100) against time t (days). The slope of the regression equation is the degradation rate constant k (d). -1 Degradation half-life t 1 / 2 = ln2 / k, in days (d), each sample was repeated 3 times.

[0074] The test results are shown in Table 2.

[0075]

[0076] As shown in Table 2, the composite wound dressing prepared by the present invention has excellent compressive strength of the inner functional layer, and the degradation half-life of the outer support layer and the inner functional layer can be matched by process control, so that the degradation rates of the inner and outer layers are similar, avoiding the problem of the outer layer degrading too early and losing support or the inner layer remaining and not being able to be absorbed synchronously.

[0077] Comparative Example 1: Carvacrol was only physically mixed without chemical grafting. Therefore, carvacrol was easily lost prematurely during subsequent dialysis and compounding processes, failing to maintain a stable and long-lasting antibacterial effect. Furthermore, there was no significant difference in compressive strength, indicating that carvacrol grafting had little impact on the dressing's mechanical properties. Comparative Example 2: Due to the lack of secondary cross-linking with genipin, the inner layer collagen sponge had insufficient cross-linking, resulting in a significantly accelerated degradation rate. The outer layer, without acetylation treatment, degraded even slower, ultimately leading to a degradation half-life ratio of 8.09:1 between the inner and outer layers, indicating extremely poor matching and inability to meet the requirement of simultaneous degradation. Comparative Example 3: The inner layer did not use woven fabric as a reinforcing skeleton, resulting in a compressive strength of only 87.4 kPa, far lower than the embodiments of this invention, indicating insufficient mechanical support performance. Comparative Example 4: The outer ramie layer did not undergo multiple modification treatments, resulting in a slower fiber degradation rate than modified ramie, with a degradation half-life ratio increasing to 2.36:1. The overall mechanical strength of the outer layer also decreased.

[0078] (3) Antibacterial test of inner dressing: The composite wound dressing was cut to a diameter of 10 mm as Sample 1; the inner functional layer of the grafted carvacrol collagen sponge was used as Sample 2. A piece of ramie fabric, identical in size to the outer support layer of the composite wound dressing but without antibacterial treatment, was taken as Control Sample 1. The ungrafted collagen solution was directly freeze-dried to obtain the ungrafted collagen sponge, which served as Control Sample 2. For experimental rigor, samples should not be sterilized.

[0079] Inoculation with bacteria: Prepare a sterile culture medium. Pour 10 mL of agar medium into a sterile petri dish and allow it to solidify. Prepare another petri dish as the inoculation medium. Take 150 mL of 45°C agar medium and place it in a flask, then add 1 mL of the experimental bacterial suspension. Using sterilized forceps, place the sample and control sample in the center of the petri dish, pressing them gently and evenly onto the agar medium until they are in good contact. After placing the sample on the agar medium, immediately incubate at 37°C for 14–24 hours, ensuring good contact between the sample and the agar medium throughout the incubation period. The condition of the sample and control sample in the petri dish after 14 hours of bacterial incubation.

[0080] The test results are shown in Table 3.

[0081] Table 3 Results of antibacterial test on inner dressing

[0082] As shown in Table 3, this invention, through chemical grafting, binds carvacrol to collagen molecules, stably preserving the antibacterial active ingredients. Both the individual inner functional layer and the final composite dressing exhibit stable antibacterial effects. Ungrafted pure collagen sponge itself has no antibacterial ability, and the original ramie fabric itself does not produce bacterial growth, meeting the basic requirements for medical materials. After grafting, the antibacterial components are not lost due to subsequent processing steps, thus stably maintaining the antibacterial environment of the wound and reducing the risk of wound infection.

[0083] (4) Wound healing rate determination: Healthy male SD rats (weight 200±20 g, 6-8 weeks old) were selected and acclimatized for 7 days in a standard SPF animal room (temperature 22±2℃, humidity 50%~60%, 12h light / 12h dark cycle) before the experiment. Before the operation, the animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 50 mg / kg. The hair on the back was thoroughly shaved with an animal shaver and then depilatory cream was applied. The surgical area was disinfected alternately with povidone-iodine and 75% ethanol. Four wounds were marked on both sides of the spine on the back (avoiding the scapula and pelvic bone areas) with a circular skin punch with a diameter of 2.0 cm. The full-thickness skin was removed along the marked lines to the deep fascia layer with sterile ophthalmic scissors and forceps to form a full-thickness skin defect. After the wound was formed, the dressing samples with different group numbers were immediately covered on the wound, covered with sterile gauze and fixed in a ring with medical breathable tape. The dressing was changed every 2 days after the operation. The wounds were photographed using a digital camera (including a scale reference) under the same magnification and light source conditions on postoperative days 0 (immediately after modeling), 7, and 14. Wound healing rate (%) = (initial wound area - current wound area) / initial wound area × 100%, where the initial wound area was based on the wound area in the postoperative day 0 photograph. Four parallel wounds (symmetrical left and right) were set up for each rat and each test sample, with six rats per group (n=24). Results are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0084] The test results are shown in Table 4.

[0085] Table 4 Test results of Examples 1-4 and Comparative Examples 1-4

[0086] As shown in Table 4, the wound healing rate of the composite wound dressing prepared by this invention was around 90% 14 days post-surgery, significantly higher than all comparative examples, with statistically significant differences. Example 1 showed the highest healing rate at 94.6%, demonstrating that the process parameters designed in this invention can maximize wound healing. Comparative Example 1, due to the physical mixing of carvacrol, experienced premature loss of antibacterial components during processing, resulting in an inability to maintain a stable antibacterial environment on the wound surface, leading to a healing rate of only 74.8%, significantly increasing the risk of infection and delaying the healing process. Comparative Example 2 exhibited poor degradation matching between the inner and outer layers; the inner layer degraded prematurely, failing to maintain a moist repair environment, while long-term residue in the outer layer easily affected the growth of new tissue, resulting in a healing rate of only 78.6%. Comparative Example 3 lacked a woven skeleton for support, resulting in insufficient mechanical properties and an inability to provide a stable repair space for the wound, with a healing effect far lower than that of the examples in this invention. Comparative Example 4, with its unmodified outer layer, had a slow degradation rate and insufficient mechanical support, ultimately achieving a healing rate of only 73.5%, verifying the positive effects of fiber modification and structural design on wound healing in this invention.

[0087] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and function as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A composite wound dressing, characterized in that, It includes an outer support layer and an inner functional layer; The outer support layer is a modified ramie fiber nonwoven fabric, and the modification treatment includes low-temperature plasma treatment and acetylation modification of the ramie fibers; The inner functional layer is a composite of ramie woven fabric and low-antigen collagen sponge grafted with carvacrol. The collagen sponge penetrates through the gaps and pores of the yarn in the woven fabric, forming a structure in which the collagen sponge and ramie fibers wrap around and interweave with each other.

2. The composite wound dressing according to claim 1, characterized in that, The low-antigen collagen contains residual calcium ions; The carvacrol is covalently grafted onto the carboxyl group of the collagen via an esterification reaction; The calcium ions and carvacrol form a synergistic antibacterial effect in the slightly acidic environment of the wound.

3. The composite wound dressing according to claim 1, characterized in that, The low-temperature plasma treatment is carried out in an oxygen or nitrogen atmosphere, with a treatment power of 100~300W and a treatment time of 2~10min.

4. The composite wound dressing according to claim 1, characterized in that, The raw material for extracting the low-antigen collagen is scraps of leather from tanning and liming processes.

5. The composite wound dressing according to claim 1, characterized in that, The inner functional layer undergoes a secondary cross-linking treatment with a cross-linking agent, the amount of which is 0.10% to 0.35% of the dry weight of the collagen.

6. A method for preparing a composite wound dressing as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Ramie fibers are subjected to enzymatic degumming treatment, steam explosion treatment, low-temperature plasma treatment and acetylation modification in sequence to obtain modified ramie fibers. The modified ramie fibers are formed as an outer support layer. At the same time, a portion of the modified ramie fibers are woven into a woven fabric. S2: Using leather scraps from tanning and liming as raw materials, the residual calcium ion content in the raw materials is controlled at 0.8%~1.2% of the dry weight through deliming treatment. Then, the raw materials are extracted, purified and dialyzed by alkaline protease to obtain a low antigen collagen solution. S3: In the presence of a crosslinking agent, carvacrol is covalently grafted onto the carboxyl group of the low-antigen collagen to obtain a carvacrol-grafted collagen solution. S4: The collagen solution grafted with carvacrol obtained in step S3 is filled into the woven fabric obtained in step S1. After vacuum degassing, it is freeze-dried using a freeze-drying process. After drying, a composite of collagen sponge and ramie three-dimensional skeleton interpenetrates, which serves as the inner functional layer. S5: Combine the inner functional layer obtained in step S4 with the outer support layer obtained in step S1, and sterilize to obtain the composite wound dressing.

7. The method for preparing the composite wound dressing according to claim 6, characterized in that, The deashing treatment in step S2 includes: first treating the scraps of the ash-soaked skin with a 1.0%~2.5% ammonium sulfate solution for 10~30 min, then treating them with a buffer solution containing 0.3%~0.8% sodium citrate and 0.05~0.15 mol / L calcium chloride for 20~60 min, monitoring the endpoint with a calcium ion selective electrode or EDTA titration method, so that the residual calcium ion content in the raw material is controlled at 0.8%~1.2% of the dry weight; And / or, the parameters for alkaline protease hydrolysis extraction are: enzyme addition amount of 8%~15% of the dry weight of raw material, temperature of 33~40℃, pH of 8.5~9.5, and time of 4~6 hours.

8. The method for preparing the composite wound dressing according to claim 6, characterized in that, The freeze-drying process in step S4 includes: Pre-freezing stage: Pre-freeze at -20~-30℃ for 2~4 hours; Deep freezing stage: Cool to -40~-60℃ at a rate of 2~5℃ / h and deep freeze for 4~8 hours; Gradient heating: Gradually increase the temperature to 20℃~25℃ at a heating rate of 1~3℃ / h, with a vacuum degree ≤20 Pa.

9. The method for preparing the composite wound dressing according to claim 6, characterized in that, In step S1, the acetylation modification uses acetic anhydride as the acetylation agent and reacts at 50-70°C for 30-90 min in the presence of a catalyst.

10. The method for preparing the composite wound dressing according to claim 6, characterized in that, In step S3, the crosslinking agent used for the carvacrol grafting reaction is EDC / NHS, the reaction temperature is 25~37℃, and the reaction time is 4~12 hours. And / or, in step S3, the collagen solution grafted with carvacrol undergoes a secondary cross-linking treatment with a cross-linking agent before step S4. The cross-linking agent used in the secondary cross-linking treatment is genipin, with an addition amount of 0.10%~0.35% of the dry weight of collagen, a reaction temperature of 20~30℃, and a reaction time of 4~8 hours.

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