Artificial dermis dressing as well as preparation method and application thereof

Abstract

The invention relates to the field of biological dressings, and discloses an artificial dermis dressing as well as a preparation method and application thereof. The preparation method comprises the following steps: adding water into a container, and freezing until the water is frozen; then adding a collagen A solution, freezing and standing; continuing to add collagen B liquid, freezing and standing; continuing to add collagen A liquid, freezing and then freeze-drying; and cross-linking in a glutaraldehyde solution, and cleaning to obtain the artificial dermis dressing. The dressing provided by the invention can provide a good stent effect and promote cell migration and proliferation, and the healing rate is close to 100% after wound making. In the wound healing process, the dressing can inhibit collagen deposition, relieve inflammatory response, inhibit abnormal hyperplasia of blood vessels, inhibit excessive proliferation of fibroblasts and excessive formation of granulation tissues, and effectively prevent scar formation. The dressing can also effectively inhibit abnormal vascular hyperplasia in a wound area, and fibrosis and scar hyperplasia caused by excessive vascular hyperplasia are prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of biological dressings, and more particularly to an artificial dermal dressing and a preparation method and application thereof. Background Art

[0002] The skin, the largest organ in the human body, serves as the first line of defense against numerous environmental stimuli, protecting the body from pathogens, ultraviolet rays, and other pollutants. It not only regulates body temperature and water balance but also plays a key role in regulating the body's immune response. However, trauma, burns, or other injuries can severely disrupt the structure and function of the skin, leading to tissue damage. Skin damage not only triggers inflammation and infection but can also delay or impair wound healing, including scar formation. Scar formation is primarily caused by the loss of the dermis, which leads to excessive fibroblast proliferation and the resulting thick, disorganized arrangement of collagen fibers. The wound healing process typically triggers a series of pathological changes, including the activation of an inflammatory response, excessive collagen deposition, abnormal vascular proliferation, excessive fibroblast activation and proliferation, and excessive granulation tissue formation, ultimately leading to abnormal thickening of the skin at the healing site and the formation of a scar. Scarring not only affects the appearance and function of the skin but can also cause pain, itching, and psychological distress. Therefore, inhibiting scar formation is crucial for promoting wound healing and restoring skin function.

[0003] As a biomedical material, artificial dermal dressings have made significant progress in the field of skin wound healing in recent years. These dressings are typically made from biocompatible materials such as collagen and hyaluronic acid, and can mimic the structure and function of natural dermis. Artificial dermal dressings accelerate wound healing by providing a temporary scaffold that supports cell proliferation and differentiation, promotes angiogenesis and collagen deposition, and ultimately accelerates wound healing. Furthermore, these dressings can effectively reduce the risk of infection and scar formation, leading to their widespread application in wound repair for large burns, chronic ulcers, and post-surgical procedures.

[0004] Currently, the design of artificial dermal dressings is often based on a homogeneous structure with uniform pore size. However, the structure of the human dermis is not uniform at different depths, both in terms of gap size and tissue structure. The article "Biomimeticfibroblast-loaded artificial dermis with "sandwich" structure and designedgradient pore sizes promotes wound healing by favoring granulation tissueformation tissue formation and wound re-epithelialization" discloses a fibroblast-loaded artificial dermis composed of three layers of scaffolds with different pore sizes. The outer layer has relatively large pores, and the middle layer has smaller pores. By loading fibroblasts, the artificial dermis promotes granulation tissue formation and wound re-epithelialization to promote wound healing. However, the above-mentioned artificial dermal dressing has insufficient support and is easily separated from the tissue wound surface. It is not conducive to maintaining a moist environment in open wounds and does not have the function of inhibiting scar formation. Summary of the Invention

[0005] The present invention provides an artificial dermal dressing to overcome the defects of the prior art, such as insufficient support, easy separation from the tissue wound surface, poor maintenance of the moist environment of the open wound, and inability to inhibit scar formation.

[0006] Another object of the present invention is to provide a method for preparing an artificial dermal dressing;

[0007] Another object of the present invention is to provide an application of an artificial dermal dressing.

[0008] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0009] A method for preparing an artificial dermal dressing comprises the following steps:

[0010] S1: Freeze the water in the container until it freezes;

[0011] S2: Collagen A solution is then added and allowed to stand after freezing;

[0012] S3: Continue to add collagen B solution, freeze and place;

[0013] S4: Continue to add collagen A solution, freeze and then freeze-dry;

[0014] S5: cross-linking in glutaraldehyde solution and washing to obtain artificial dermal dressing;

[0015] The mass concentration of the collagen A solution is 0.30-0.35%, and the mass concentration of the collagen B solution is 2.20-2.30%.

[0016] Preferably, the collagen A solution and the collagen B solution are type I collagen solutions.

[0017] Preferably, the type I collagen is derived from pig skin.

[0018] Pigskin has a high similarity to human skin. The dressing prepared using pigskin collagen in the present invention has good biocompatibility and relatively low immunogenicity, which reduces the risk of immune rejection during use. Rat collagen or other sources are quite different from human collagen and may trigger a stronger immune response, with a higher likelihood of immune rejection. In addition, pigskin is abundant and easy to obtain, especially on an industrial scale, and has significant cost advantages. Pigskin collagen extraction technology is mature and suitable for large-scale production. Collagen from sources such as rat tail tendons is difficult to extract and has a low yield, so the cost is high and is usually limited to small-scale experimental studies.

[0019] Preferably, the mass concentration of the collagen A solution is 0.34%, and the mass concentration of the collagen B solution is 2.27%.

[0020] Furthermore, the added amounts of collagen A solution in S2, collagen B solution in S3, and collagen A solution in S4 are the same.

[0021] Preferably, the thickness of the obtained artificial dermal dressing is 0.8 to 1.5 μm.

[0022] Preferably, the thickness of the obtained artificial dermal dressing is 0.877-1.433 μm.

[0023] Furthermore, the freezing conditions in S2 to S4 are -55 to -65°C for 0.5 to 1 hour.

[0024] Preferably, the freezing condition in S2 to S4 is -60°C for 1 hour.

[0025] Furthermore, the storage condition in S2-S3 is 25-27° C. for 10-15 minutes.

[0026] Preferably, the storage condition in S2-S3 is 25-27° C. for 15 minutes.

[0027] Furthermore, the mass concentration of the glutaraldehyde solution in S5 is 0.2% to 0.25%.

[0028] Preferably, the mass concentration of the glutaraldehyde solution in S5 is 0.25%.

[0029] Furthermore, the cross-linking condition in S6 is cross-linking at 25-27° C. for 45-50 hours.

[0030] Preferably, the cross-linking condition in S6 is cross-linking at 25-27° C. for 48 hours.

[0031] An artificial dermal dressing is prepared by the artificial dermal dressing preparation method.

[0032] Furthermore, the artificial dermal dressing has a three-layer structure, the pore diameters of the first and third layers are 150 to 550 μm, and the pore diameter of the second layer is 50 to 150 μm.

[0033] Preferably, the average long-diameter pore diameter of the first and third layer structures is 530.91 μm, the average short-diameter pore diameter is 133.43 μm, and the average pore diameter of the second layer structure is 82.32 μm.

[0034] Furthermore, the artificial dermal dressing has a three-layer structure, the porosity of the first and third layers is 75% to 85%, and the porosity of the second layer is 55% to 70%.

[0035] An application of the artificial dermal dressing is to prepare a dressing for inhibiting scar formation.

[0036] The artificial dermal dressing of the present invention is composed of three layers of collagen scaffolds with different pore sizes, similar to a sandwich structure, which mimics the natural structure of the human dermis. The outer layer has a relatively large pore size, and the middle layer has a smaller pore size. The relatively high mass concentration of collagen solution forms a denser pore size, which not only provides stronger support for the wound surface, but also provides more abundant adhesion sites for cells, promoting cell penetration and crawling into the dressing. Experiments have shown that the artificial dermal dressing of the present invention can inhibit collagen deposition, reduce inflammatory reactions, inhibit abnormal vascular proliferation, inhibit excessive proliferation of fibroblasts and excessive formation of granulation tissue during the wound healing process, thereby effectively preventing scar formation.

[0037] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0038] 1. Significant wound healing: The artificial dermis dressing of the present invention provides an excellent scaffolding effect, promoting cell migration and proliferation. The healing rate of the artificial dermis dressing of the present invention increased rapidly at days 7 and 14 after wound initiation, ultimately approaching 100%. Compared with conventional double-layer artificial dermis dressings, the artificial dermis dressing of the present invention presents a smoother skin surface after wound healing, helping to reduce scar formation.

[0039] 2. Inhibit scar formation: ① Inhibit abnormal thickening of the skin. On the 7th to 14th day after wound creation, compared with the abnormal proliferation of the skin of the conventional double-layer artificial dermal dressing, the skin thickness of the artificial dermal dressing of the present invention is close to that of normal skin, and there is no obvious epidermal thickening. ② Reduce collagen deposition. The dermis layer of the conventional double-layer artificial dermal dressing is mainly composed of type III collagen and has a relatively loose structure, while the dermis layer of the artificial dermal dressing of the present invention has a large amount of thick type I collagen fibers accumulated in the healing part, which helps to maintain skin elasticity and normal function, thereby reducing scar formation. ③ Inhibit excessive proliferation and activation of fibroblasts. The artificial dermal dressing of the present invention showed fewer PCNA-positive cells on Day 7 and Day 14, indicating that it effectively inhibited the excessive proliferation of fibroblasts. In addition, it can also inhibit the excessive transformation of fibroblasts into myofibroblasts, further inhibiting the formation of fibrosis and scars.

[0040] 3. Reduce inflammatory response and abnormal vascular proliferation: The artificial dermal dressing of the present invention significantly reduces the expression of the inflammatory factor TNFα in the early healing stage, alleviates the inflammatory response, avoids the negative impact of excessive inflammation on the healing process, and provides a better healing environment for the wound. In addition, when the angiogenesis on Day 7 was detected, the CD31 positive area of ​​the artificial dermal dressing of the present invention (2.399±0.2167%) was significantly smaller than that of the conventional double-layer artificial dermal dressing (0.5906±0.111%). This shows that the artificial dermal dressing of the present invention can effectively inhibit the abnormal vascular proliferation in the wound area, thereby preventing fibrosis and scar hyperplasia caused by excessive vascular proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 These are photos of artificial dermal dressings, A is a conventional double-layer artificial dermal repair material (Dressing 1); B is the artificial dermal dressing of Example 1 of the present invention (Dressing 2).

[0042] Figure 2 To establish a full-thickness skin defect model on the back of mice, a circular full-thickness skin defect wound with a diameter of 8 mm was established on the left and right sides of the back of C57 mice. The artificial dermis material of this group was implanted in the wound on the left and right sides of the mice according to the grouping situation.

[0043] Figure 3 Figure 3 shows the effects of two artificial dermal dressings on wound healing. A is a diagram of the in vivo wound repair process; B is a quantitative analysis of the wound healing rate (n=6, ns, no statistical significance).

[0044] Figure 4 HE staining of skin tissue at various time points during wound healing (the scale in the lower left corner is 1 mm).

[0045] Figure 5Figure 2: Cell migration in the artificial dermal dressing of the Dressing 2 group at Day 2. A shows DAPI staining of cells in the inner area; B shows DAPI staining of cells at the upper edge (the scale bar in the lower right corner is 50 μm).

[0046] Figure 6 The results of epidermal and dermal thickness measurements at the wound healing site on Day 14. A is a schematic diagram of dermal thickness measurement (normal dermis, Dressing 1 group, and Dressing 2 group); B is a statistical analysis chart of dermal thickness; C is a schematic diagram of epidermal thickness measurement (normal epidermis, Dressing 1 group, and Dressing 2 group); D is a statistical analysis chart of epidermal thickness, ****P < 0.0001, ***P < 0.001, **P < 0.01.

[0047] Figure 7 These are the Masson staining results of the wound healing sites in different material groups on Day 14 (the scale bars in the lower left corner are 1 mm and 100 μm, respectively).

[0048] Figure 8 The results of picrosirius red staining of the wound healing sites in different material groups on Day 14 (the scale bars in the lower right corner are 500 μm and 100 μm respectively).

[0049] Figure 9 Figure 2: TNFα staining results of skin tissue on Day 2. A is the comparison of TNFα staining of skin tissue between Dressing 1 and Dressing 2 groups; B is the statistical analysis of the percentage of TNFα-positive area, **P < 0.01.

[0050] Figure 10 Figure 3: CD31 fluorescence staining results of wound healing sites on Day 7. (A) Comparison of CD31 fluorescence staining of wound healing sites between Dressing 1 and Dressing 2 groups. (B) Statistical analysis of CD31 fluorescence area percentage, ****P < 0.0001.

[0051] Figure 11 Figure 3: PCNA fluorescence staining results of wound healing sites on Day 7. A is a comparison of PCNA fluorescence staining of wound healing sites between Dressing 1 and Dressing 2 groups. B is a statistical analysis of the number of PCNA-positive cells, ****P < 0.0001.

[0052] Figure 12 Figure 2 shows the results of PCNA fluorescence staining at the wound healing site on Day 14. A shows the comparison of PCNA fluorescence staining at the wound healing site between Dressing 1 and Dressing 2 groups. B shows the statistical analysis of the number of PCNA-positive cells. ***P < 0.001.

[0053] Figure 13 Figure 14: α-SMA fluorescence staining results at the wound healing site on Day 14. A is a comparison of α-SMA fluorescence staining at the wound healing site between the Dressing 1 group and the Dressing 2 group. B is a statistical analysis of the relative mean fluorescence intensity of α-SMA, **P < 0.01.

[0054] Figure 14 These are scanning electron micrographs of artificial dermal dressings, where A is a single-layer material prepared with collagen A solution of the present invention, and B is a single-layer material prepared with collagen B solution;

[0055] Figure 15 Statistical analysis of the epidermal and dermal thickness measurements at the wound healing site on Day 14. A is the statistical analysis chart of epidermal skin thickness; B is the statistical analysis chart of dermal skin thickness. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0057] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0058] Example 1 (Dressing 2)

[0059] The type I collagen solution contains liquid A (mass concentration of 0.34%) and liquid B (mass concentration of 2.27%). Type I collagen is derived from pig skin. Take a 12-well plate, add 1.5mL of water to each well, freeze at -60℃ for 10min, make the water surface flat and frozen, add 300μL of liquid A to each well, cast flat, freeze at -60℃ for 1h, take out and place at 26℃ for 15min. Add 300μL of liquid B, cast flat, freeze at -60℃ for 1h, take out and place at 26℃ for 15min. Add 300μL of liquid A again, cast flat, freeze at -60℃ for 1h, and put into a freeze dryer for freeze drying for 24h. The freeze-dried sample is cross-linked in 0.25% glutaraldehyde solution at 26℃ for 48h. Finally, wash with purified water 5 times and sterilize for use.

[0060] Example 2

[0061] The type I collagen solution contains collagen A solution (mass concentration of 0.30%) and collagen B solution (mass concentration of 2.20%). Type I collagen is derived from pig skin. Take a 12-well plate, add 1.5mL of water to each well, freeze at -60℃ for 10 minutes, make the water surface flat and frozen, add 300μL of collagen A solution to each well, cast flat, freeze at -65℃ for 1 hour, take out and let it stand at 25℃ for 15 minutes. Add 300μL of collagen B solution, cast flat, freeze at -65℃ for 1 hour, take out and let it stand at 25℃ for 15 minutes. Add 300μL of collagen A solution again, cast flat, freeze at -65℃ for 1 hour, and put it in a freeze dryer for freeze drying for 24 hours. The freeze-dried sample is cross-linked in 0.20% glutaraldehyde solution at 27℃ for 50 hours. Finally, wash it with purified water 5 times and sterilize it for use.

[0062] Example 3

[0063] The type I collagen solution contains collagen A solution (mass concentration of 0.35%) and collagen B solution (mass concentration of 2.30%). Type I collagen is derived from pig skin. Take a 12-well plate, add 1.5mL of water to each well, freeze at -60℃ for 10 minutes, make the water surface flat and frozen, add 300μL of collagen A solution to each well, cast flat, freeze at -55℃ for 0.5h, take out and let it stand at 27℃ for 10min. Add 300μL of collagen B solution, cast flat, freeze at -55℃ for 0.5h, take out and let it stand at 27℃ for 10min. Add 300μL of collagen A solution again, cast flat, freeze at -55℃ for 0.5h, and put it in a freeze dryer for freeze drying for 24h. The freeze-dried sample is cross-linked in 0.25% glutaraldehyde solution at 25℃ for 45h. Finally, wash it with purified water 5 times and sterilize it for use.

[0064] Comparative Example 1 (Dressing 1)

[0065] First, type I collagen and chondroitin sulfate were mixed and stirred thoroughly to form a homogeneous solution. The mixed solution was poured into a mold and frozen at -80°C for 4 hours. Subsequently, the frozen sample was placed in a freeze dryer and freeze-dried at -50°C for 48 hours to obtain a dry porous collagen scaffold. Next, the scaffold was placed in a 25% glutaraldehyde solution and cross-linked at 37°C under closed conditions for 6 hours. Neutralized with a 0.1M glycine aqueous solution for 48 hours to remove free glutaraldehyde, and then rinsed with deionized water 5 times. Finally, the treated collagen scaffold was compounded with a layer of medical silicone membrane by physical pressing to obtain a conventional double-layer artificial dermis repair material.

[0066] Comparative Example 2

[0067] Collagen solutions from SD rat tail tendons at mass concentrations of 0.26% and 0.13% were transferred to individual wells of a 12-well plate using a pipette. The collagen solution was frozen at -80°C for 4 hours and then freeze-dried in a SuperModulyo freeze dryer for 48 hours. The freeze-dried scaffold was placed in a 25% glutaraldehyde aqueous solution and cross-linked in a sealed box at 37°C for 6 hours. The construct was soaked in a 0.1M glycine aqueous solution for 48 hours, then washed five times with deionized water and sterilized for use. The prepared porous collagen scaffolds of different pore sizes were stacked before use.

[0068] Comparative Example 3

[0069] Use a pipette to transfer type I collagen solutions A (mass concentration of 0.34%) and B (mass concentration of 2.27%) derived from pig skin to each well of a 12-well plate. Freeze at -80°C for 4 hours and then freeze-dry in a Super Modulyo freeze dryer for 48 hours. The freeze-dried samples are cross-linked in a 0.25% glutaraldehyde solution at 26°C for 48 hours. Finally, wash with purified water five times and sterilize before use. The prepared porous collagen scaffolds of different pore sizes are stacked before use.

[0070] Verification Method

[0071] (1) Animal experiment verification

[0072] After C57 mice were divided into groups, a full-length skin defect wound was established on the back of the mice ( Figure 2 ), artificial dermal dressings from each example and comparative example were implanted, and wound surfaces were observed and data collected at D0, D2, D7, and D14 after wound initiation. Wound surface area was measured using ImageJ software at D0, D2, D7, and D14, and wound surface area data for the different material groups at each time point were obtained, and healing rates were calculated.

[0073] (2) Effect of artificial dermal dressing on skin wound healing

[0074] Skin tissue from wounds implanted with a conventional double-layer artificial dermis repair material (Comparative Example 1) and the artificial dermis dressing of the present invention was collected on Day 2, Day 7, and Day 14 for HE, Masson's, picrosirius red, IHC, and IF staining. Histological analysis revealed changes in skin tissue, collagen deposition, expression of inflammatory factors, angiogenesis, and cell proliferation during wound healing.

[0075] (3) Strain recovery rate

[0076] The dressing strip was stretched to 150% of its original length, held for 30 seconds, and then released. The recovered length was measured and the recovery rate was calculated.

[0077]

[0078] Among them, L0 is the initial length, L1 is the length after stretching, and L2 is the length restored after release.

[0079] Result Analysis

[0080] The process of wound healing and scar formation typically triggers a series of pathological changes, including activation of the inflammatory response, extensive collagen deposition, abnormal vascular proliferation, overactivation and proliferation of fibroblasts, and excessive formation of granulation tissue, ultimately leading to abnormal thickening of the skin at the healing site and the formation of a scar. The artificial dermal dressing of the present invention can effectively alleviate the inflammatory response during the healing process, inhibit collagen deposition and abnormal vascular proliferation, inhibit overactivation of fibroblasts and excessive formation of granulation tissue, and inhibit abnormal thickening of the skin, thereby significantly reducing the risk of scar formation.

[0081] 1. Compared with Comparative Example 1

[0082] (1) Good effect in promoting wound healing

[0083] Figure 1 A is a conventional double-layer artificial leather repair material (Dressing 1), Figure 1 The difference in appearance of the artificial dermal dressing (Dressing 2) of Example 1 of B is that the conventional double-layer artificial dermal repair material is thinner and more transparent.

[0084] Figure 3 Figure B shows a quantitative analysis of wound healing rates for the two dressings at different time points. As can be seen, the healing rates for Dressing 1 and Dressing 2 were similar at Day 2, with no significant difference between the two. The healing rates increased rapidly at Days 7 and 14, ultimately approaching 100%. Throughout the healing process, there was no significant difference between Dressing 1 and Dressing 2, demonstrating that both dressings were equally effective in promoting wound healing.

[0085] from Figure 3 As shown in Figure A, the wound areas of Dressing 2 and Dressing 1 gradually shrank over time, especially at D7 and D14, when the wounds were close to healing. Moreover, the wound surface of Dressing 2 was smoother after healing, which was more conducive to reducing scar formation.

[0086] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0087] (2) Inhibit abnormal skin thickening

[0088] The artificial dermal dressing of the present invention and the conventional double-layer artificial dermal repair material are both imitation artificial dermal materials, which have certain pores to facilitate cell crawling and migration. The HE staining results show that the repair material of the Dressing1 group has been separated from the tissue wound site on Day 2 after the wound is made, indicating that the repair material does not play a good dermal scaffold role, and only acts as an ordinary dressing to isolate the wound from the outside world; while the artificial dermal dressing of the Dressing2 group provides a good scaffold role, and cells have crawled into the inside of the scaffold. On Day 7 after the wound is made, the skin of the Dressing1 group is significantly thickened, while the thickness of the skin of the Dressing2 group is not much different from the thickness of the normal skin on both sides; on Day 14, the wounds of the two groups of mice are completely healed, and the skin of the wound healing site of the Dressing1 group is thicker than that of the Dressing2 group, and the epidermis is significantly thickened ( Figure 4 The above phenomena all indicate that the artificial dermal dressing of the present invention can inhibit the formation of scars.

[0089] The results of DAPI staining of Day 2 tissue sections in the Dressing 2 group showed that cells had crawled into the interior of the artificial dermal scaffold material in the Dressing 2 group ( Figure 5 A) and the upper edge of the bracket ( Figure 5 B), indicating that the artificial dermal scaffold of the present invention not only provides a good scaffolding effect, but also enables cells to crawl and migrate normally, which is conducive to the healing of skin wounds.

[0090] The dermal thickness of the wound healing site on Day 14 was measured. The results showed that the dermal thickness of the Dressing 1 group was significantly increased compared with that of the normal mice ( Figure 6 A, B). There was no significant difference in dermal thickness between the Dressing2 group and normal skin, nor was there a significant difference in dermal thickness between the Dressing1 and Dressing2 groups. Epidermal measurements showed that compared with the epidermal thickness of normal mouse skin, the Dressing1 group had a significant increase in epidermal thickness, while the Dressing2 group had a smaller increase in epidermal thickness. Observing overall skin thickness, the healing site in the Dressing1 group had abnormally thickened skin, while the thickness of the healing site in the Dressing2 group was closer to normal skin ( Figure 6 C, D).

[0091] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0092] (3) Inhibit collagen accumulation

[0093] Masson staining of Day 14 tissue sections showed that a large number of blue-stained collagen fibers were visible in the dermis of the Dressing 1 group. The collagen was thick and densely arranged, and its direction was roughly parallel to the epidermis or relatively disordered. In the Dressing 2 group, red staining was mostly seen in the dermis, and the collagen arrangement structure was relatively loose ( Figure 7 This phenomenon suggests that a large amount of collagen deposition occurred in the Dressing 1 group, which easily led to skin scar hyperplasia, while the artificial dermal dressing in the Dressing 2 group may have inhibited collagen accumulation and prevented scar hyperplasia.

[0094] In normal skin tissue, type I collagen is relatively coarse and constitutes the main body of the skin, while type III collagen is fine and is the main component of the reticular structure in the skin. In scar tissue, coarse type I collagen fibers are the material basis of scar tissue fibrosis. Their large appearance makes the structure of the scar completely different from normal skin tissue, with a harder texture and no elasticity. Therefore, in the healing process, inhibiting the excessive proliferation of type I collagen and promoting the synthesis of type III collagen will help reduce scar proliferation and improve the quality of skin healing. In order to evaluate the effects of the two repair materials on type I and type III collagen during wound healing, the Day 14 tissue sections were stained with picrosirius red and imaged and analyzed using a polarized light microscope. The results showed that the healing site of the Dressing 1 group showed a large number of orange-red fibers, which were thicker and densely arranged, and were type I collagen fibers; the healing site of the Dressing 2 group showed green fine fibers, which were mainly type III collagen ( Figure 8 This phenomenon indicates that the artificial dermal scaffold in the Dressing 2 group has a certain inhibitory effect on the excessive proliferation of type I collagen, which helps to reduce scar hyperplasia and improve the quality of wound healing.

[0095] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0096] (4) Reduce inflammatory response

[0097] Skin damage triggers a series of inflammatory responses. The inflammatory phase of wound healing is a rapid and highly coordinated process. Excessive inflammatory responses can greatly affect wound healing. To evaluate the effects of the two repair materials on the inflammatory phase of wound healing, immunohistochemical staining was used to detect the expression of inflammatory factor TNFα at the wound margins of the Dressing 1 and Dressing 2 groups on Day 2. The results showed that on Day 2 after injury, the TNFα level at the wound margins of the Dressing 1 group was significantly higher than that of the Dressing 2 group ( Figure 9 A, B). This indicates that the artificial dermal dressing in the Dressing 2 group can alleviate the inflammatory response in the early stages of wound healing, thereby maintaining a good healing environment on the wound surface.

[0098] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0099] (5) Inhibit abnormal growth of blood vessels

[0100] In the process of skin injury repair, angiogenesis is the key to wound repair, but too many blood vessels can easily lead to scar hyperplasia. In order to evaluate the effect of the two repair materials on angiogenesis during wound healing, we detected the expression of CD31 by immunofluorescence staining to explore the state of angiogenesis in the skin wound area. The results of CD31 fluorescence staining showed that on Day 7, the percentage of CD31 fluorescence area in the Dressing 1 group and the Dressing 2 group were 2.399±0.2167% and 0.5906±0.111%, respectively, which were statistically significantly different ( Figure 10 ), indicating that the number of blood vessels in the wound healing area of ​​Dressing 1 group was greater than that of Dressing 2 group. This suggests that the artificial dermal dressing used in Dressing 2 group has a certain inhibitory effect on the abnormal increase of blood vessels during wound repair.

[0101] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0102] (6) Inhibit excessive proliferation of fibroblasts and excessive formation of granulation tissue

[0103] PCNA is a nuclear protein expressed in G1-M phase cells. It is closely related to cell DNA synthesis and plays an important role in the initiation of cell proliferation. It is an indicator reflecting the state of cell proliferation. The expression of PCNA in the wound healing site on Day 7 and Day 14 was detected by immunofluorescence staining to observe the cell proliferation in the healing site at these two time points. The results of PCNA fluorescence staining showed that on Day 7, the PCNA-positive position was mainly in the dermis of the healing site. The number of PCNA-positive cells in the Dressing 1 group was significantly higher than that in the Dressing 2 group ( Figure 11 During the skin injury repair phase, dermal fibroblasts proliferate and migrate to the injury site, forming granulation tissue to promote wound healing. However, excessive granulation tissue formation can also lead to scar formation. This result indicates that the skin tissue in the Dressing 1 group is more likely to form scars than the Dressing 2 group.

[0104] On Day 14, PCNA-positive cells were mainly concentrated in the basal layer, and the number of PCNA-positive cells in the Dressing 1 group was greater than that in the Dressing 2 group ( Figure 12During wound healing, basal cells activate, proliferate, and differentiate, which is crucial for wound re-epithelialization. The number of PCNA-positive cells in scar tissue is significantly higher than in normal skin. These cells are primarily distributed in the epidermis, with significant numbers present in the basal cell layer, stratum spinosum, and stratum granulosum. These results indicate that on Day 14, the skin tissue in the Dressing 1 group resembled scar tissue more closely than that in the Dressing 2 group. These results demonstrate that the artificial dermal dressing can inhibit excessive fibroblast proliferation and granulation tissue formation, demonstrating a limited inhibitory effect on scar hyperplasia.

[0105] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0106] (7) Inhibit excessive activation of fibroblasts

[0107] The transformation of fibroblasts into myofibroblasts is the core of tissue repair and fibrosis, and overactivation of this process will lead to fibrosis and scar formation. The expression of α-SMA is a key feature of myofibroblasts. Immunofluorescence staining was used to detect the expression of α-SMA in the wound healing site of the Dressing1 group and the Dressing2 group on Day 14. The results showed that the relative mean fluorescence intensity of α-SMA in the Dressing1 group and the Dressing2 group on Day 14 was 1.000±0.1243 and 0.7127±0.0889, respectively, which was statistically significantly different ( Figure 13 ), that is, the expression level of α-SMA in the wound healing site of the Dressing 1 group was higher than that of the Dressing 2 group. This phenomenon suggests that the artificial dermal dressing in the Dressing 2 group has a certain inhibitory effect on the excessive activation of fibroblasts.

[0108] The experimental results of Examples 2 and 3 are similar to those of Dressing 2.

[0109] 2. Compared with Comparative Example 2

[0110] The first and third porous structures of the present invention are flat ( Figure 14 A), the average pore diameter of the long diameter is 530.91μm, and the average pore diameter of the short diameter is 133.43μm, which helps to enhance the adhesion stability between the material and the wound surface; the second layer of porous structure is square and round ( Figure 14 B), with an average pore size of 82.32 μm, provides a favorable three-dimensional space to promote deep cell migration and tissue regeneration. The porosity of the first and third layers obtained in Example 1 of the present invention is 75-85%, and the porosity of the second layer is 55-70%. The relatively high concentration of collagen solution forms denser pores, which not only provide stronger support for the wound surface but also provide more abundant adhesion sites for cells, promoting cell penetration and migration into the dressing. Figure 15 It also shows that the thickness of the epidermis and dermis at the wound healing site of Comparative Example 2 on Day 14 was significantly increased compared with Example 1, and the scar formation trend was enhanced.

[0111] The porous structures of Comparative Example 2 are all nearly circular, with the pore sizes of the first and third layers being 166.9 μm, and the pore size of the second layer being 87.7 μm. The HE results of the mouse wounds in Comparative Example 2 on Day 3 are consistent with the literature (Biomimetic fibroblast-loaded artificial dermis with "sandwich" structure and designed gradient pore sizes promotes wound healing by favoring granulation tissue formation tissueformation and wound re-epithelialization). The dressing has detached from the tissue wound site and the thickness of the granulation tissue increased significantly on Day 7. The detachment of the dressing cannot provide continuous wound support and cannot provide continuous guidance for fibroblasts. It can easily cause excessive proliferation of fibroblasts and the synthesis of a large amount of collagen, leading to collagen deposition and scar formation. The significant increase in the thickness of the granulation tissue also reflects a more radical tissue proliferation reaction. While Example 1 of the present invention ( Figure 4 The bionic artificial dermal dressing of Comparative Example 2 still provided a good scaffolding effect on Day 2, with cells already crawling into the scaffold. These results indicate that the performance of the dressing of Comparative Example 2 is to promote wound healing, while the dressing of the present invention inhibits scar formation.

[0112] 3. Comparison with Comparative Example 3

[0113] In Comparative Example 3, three layers of material were prepared separately, freeze-dried separately, and then stacked in a multi-layer structure during use. In the present invention, the stacking of the three layers of material was completed during the preparation process before freeze-drying. During the stacking process, the interface of the layers was in a wet state, and the fusion effect between the different layers was good. After the three-layer structure was formed, the freeze-drying process was performed, and the interlayer bonding was more firm and stable, reducing the risk of interlayer separation or peeling during actual use. The freezing conditions in Comparative Example 3 were frozen at -80°C for 4 hours, and the strain recovery rate was 67.2%. The freezing conditions in Example 1 were multiple freezing at -60°C for 1 hour, and the strain recovery rate was 85.6%. Because the wound surface loses the protection of the dressing, it is easily exposed to the external environment and more susceptible to external mechanical stimulation, resulting in an increased risk of wound infection, which may activate more inflammatory reactions and fibroblasts, causing scar formation. The overall preparation method and freezing conditions of Example 1 make the dressing more soft and elastic, able to better fit the irregular wound surface caused by burns, improve the appearance and function of the surgical site, and are more suitable for maintaining a moist environment in long-term open wounds such as chronic ulcers, inhibiting scar formation. Figure 15 It also shows that the thickness of the epidermis and dermis at the wound healing site of Comparative Example 3 on Day 14 was significantly increased compared with Example 1, and the scar formation trend was enhanced.

[0114] In Comparative Example 3, a 25% glutaraldehyde solution was used for cross-linking at 37°C in a sealed box for 6 hours. In Example 1 of the present invention, a 0.25% glutaraldehyde solution was used for cross-linking at 26°C for 48 hours. Compared with a 25% glutaraldehyde solution, a low concentration of glutaraldehyde (0.25%) is generally more biocompatible, with less residual glutaraldehyde, thus reducing the potential risk of cytotoxicity. Compared with cross-linking conditions at 37°C, cross-linking at 26°C can better protect the native structure of the protein.

[0115] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an artificial dermal dressing, characterized in that: The following steps are involved: S1: Freeze the water in the container until it freezes; S2: Collagen A solution is then added and allowed to stand after freezing; S3: Continue to add collagen B solution, freeze and place; S4: Continue to add collagen A solution, freeze and then freeze-dry; S5: cross-linking in glutaraldehyde solution and washing to obtain artificial dermal dressing; The mass concentration of the collagen A solution is 0.30% to 0.35%, and the mass concentration of the collagen B solution is 2.20% to 2.30%.

2. The method for preparing the artificial dermal dressing according to claim 1, wherein: The added amounts of collagen A solution in S2, collagen B solution in S3, and collagen A solution in S4 are the same.

3. The method for preparing the artificial dermal dressing according to claim 1, wherein: The freezing conditions in S2 to S4 are -55 to -65°C for 0.5 to 1 hour.

4. The method for preparing the artificial dermal dressing according to claim 1, wherein: The storage condition in S2-S3 is 25-27°C for 10-15 minutes.

5. The method for preparing the artificial dermal dressing according to claim 1, wherein: The mass concentration of the glutaraldehyde solution in S5 is 0.2% to 0.25%.

6. The method for preparing the artificial dermal dressing according to claim 1, wherein: The cross-linking condition in S6 is cross-linking at 25-27° C. for 45-50 hours.

7. An artificial dermal dressing, characterized in that: The artificial dermal dressing is prepared by the preparation method of any one of claims 1 to 6.

8. The artificial dermal dressing according to claim 7, characterized in that: The artificial dermis dressing has a three-layer structure, the pore diameters of the first and third layers are 150-550 μm, and the pore diameter of the second layer is 50-150 μm.

9. The artificial dermal dressing according to claim 7, characterized in that: The artificial dermis dressing has a three-layer structure, the porosity of the first and third layers is 75% to 85%, and the porosity of the second layer is 55% to 70%.

10. An application of the artificial dermal dressing according to claim 7, characterized in that: Used to prepare dressings that inhibit scar formation.

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