Multi-layer structure skin dermis stent as well as preparation method and application thereof

Abstract

The invention discloses a multilayer-structure skin dermis stent and a preparation method and application thereof. The multilayer-structure skin dermal scaffold is of a three-layer structure and is composed of an upper material layer, a middle material layer and a lower material layer, the upper material layer, the middle material layer and the lower material layer are composed of recombinant I-type humanized collagen and recombinant III-type humanized collagen, the pore size of the upper material layer and the pore size of the lower material layer are 1.5-2.1 mm, and the pore size of the upper material layer and the pore size of the lower material layer are 1.5-2.1 mm. The pore size of the middle material layer is 400-1000 [mu] m, and the porosity of the upper material layer, the middle material layer and the lower material layer is 60-70%. The stent simulates the structure and function difference of a nipple layer and a net-shaped layer in natural dermis, has good tissue adaptability and mechanical supporting performance, and has excellent performance in the aspects of simulating a dermis tissue structure, improving biological performance, improving cell integration and promoting wound repair; meanwhile, the limitation of traditional material sources and structural design is overcome.

Description

Technical Field

[0001] The present invention relates to the fields of tissue engineering and biomedical materials, and in particular to a multi-layered skin dermal scaffold and a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in the human body, and its barrier function and tissue integrity are crucial for maintaining life. In cases of deep burns, chronic ulcers, or extensive trauma, irreversible damage to the dermis often occurs, hindering tissue repair, causing severe scarring, and even impairing functional recovery.

[0003] While traditional treatments such as autologous transplantation, allogeneic transplantation, or xenogeneic acellular dermal substitutes are widely used, they face challenges such as limited donors, immune rejection, and poor integration. In recent years, the development of tissue engineering scaffolds has provided new avenues for skin regeneration. Collagen, as the body's primary structural protein, holds broad application prospects in skin tissue engineering due to its excellent biocompatibility and biodegradability.

[0004] In recent years, recombinant humanized collagen has shown significant advantages in clinical translation due to its controllable source, high structural consistency, and lack of animal-derived contamination risk, making it a key material for dermal scaffolds. However, existing scaffolds are mostly single-layer or homogeneous structures, which struggle to mimic the complex, multi-layered structure of the natural dermis. The natural dermis consists of the papillary and reticular layers. The papillary layer provides cell attachment points and promotes angiogenesis, while the reticular layer provides support for the skin. Existing scaffolds often lack this layered design, resulting in uneven cell growth and ineffective guidance of directional cell growth, which in turn impairs tissue integration and repair. More importantly, because these scaffolds fail to mimic the hierarchical structure of natural skin, they suffer from irrational stress transfer, slowing wound healing and potentially leading to scarring, compromising repair outcomes. Furthermore, the mechanical properties and bioresponsiveness of existing scaffolds are typically fixed, making them difficult to tailor to the specific wound type and stage of repair. For example, in acute wound repair, scaffolds need to provide high mechanical strength and elasticity to support angiogenesis. However, in chronic wound repair, the scaffold's degradation rate and ability to support cell proliferation are particularly important. However, existing scaffolds are often unable to adapt to these different needs, resulting in unsatisfactory effects at different stages of treatment. Existing scaffolds also have obvious problems with cell integration. They cannot effectively promote the directional growth of cells and tissue reconstruction, and lack an effective microenvironment to support cell adhesion, growth and migration, which affects the repair effect. Many scaffolds lack biological activity, cannot actively promote angiogenesis, anti-inflammatory or antibacterial, and cannot effectively respond to post-traumatic biological responses. Therefore, the development of a multilayered scaffold based on recombinant humanized collagen to improve its mechanical properties, layered induction of cell directional growth and tissue reconstruction capabilities has become a technical problem that needs to be solved urgently. Summary of the Invention

[0005] In order to address the deficiencies in the prior art, the present invention aims to provide a multi-layered skin dermal scaffold and a preparation method and application thereof.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a multi-layer skin dermal scaffold, which is a three-layer structure and consists of an upper material layer, a middle material layer and a lower material layer. The upper material layer, the middle material layer and the lower material layer are composed of recombinant type I humanized collagen and recombinant type III humanized collagen. The pore size of the upper material layer and the lower material layer is 1.5-2.1 mm, the pore size of the middle material layer is 400-1000 μm, and the porosity of the upper material layer, the middle material layer and the lower material layer is 60%-70%.

[0008] Furthermore, the mass ratio of the recombinant humanized type I collagen to the recombinant humanized type III collagen is 1:1 to 4:1;

[0009] Preferably, the mass ratio of the recombinant humanized type I collagen to the recombinant humanized type III collagen is 3:2.

[0010] Furthermore, the cross-linking degree of the multi-layered skin dermal scaffold is 40% to 70%.

[0011] Furthermore, the pore size of the upper material layer and the lower material layer is 1.9 mm, and the pore size of the middle material layer is 700 μm.

[0012] Furthermore, the thickness of the multi-layered skin dermal scaffold is determined by the thickness of the animal dermis layer, and the thicknesses of the upper material layer, the middle material layer and the lower material layer are equal or unequal.

[0013] Furthermore, the recombinant humanized type I collagen and the recombinant humanized type III collagen are modified by one or more chemical or physical methods;

[0014] Preferably, the modification methods include but are not limited to methacrylation, EDC / NHS cross-linking, photosensitive cross-linking, enzyme cross-linking or functional group grafting.

[0015] Furthermore, the scaffold further comprises one or more active ingredients that promote tissue repair to enhance the biological functionality and therapeutic effect of the scaffold;

[0016] Preferably, the active ingredients that promote tissue repair include but are not limited to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), fibronectin, anti-inflammatory drugs (such as dexamethasone, indomethacin), antimicrobial peptides or anti-infective ingredients (such as LL-37, silver ions, polymyxin B) and small molecule regeneration regulators (such as retinoic acid, antioxidants, etc.);

[0017] Preferably, the active ingredient that promotes tissue repair is loaded into the scaffold by physical adsorption, blending, cross-linking or sustained-release microcarriers, which can be combined with the porous structure of the scaffold to achieve a sustained-release function, thereby achieving continuous and stable drug release locally on the wound surface, synergistically regulating local inflammatory responses, inhibiting infection, promoting angiogenesis and tissue regeneration, and effectively improving wound healing efficiency and repair quality.

[0018] Preferably, the sustained-release microcarrier comprises microspheres, nanoparticles, liposomes or hydrogel particles;

[0019] Preferably, the scaffold further comprises a functional component;

[0020] Preferably, the functional components include but are not limited to pro-angiogenic factors, plasmid DNA, mRNA or siRNA expressing specific growth factors, which serve as a local gene transfection platform to promote cells to express repair factors in vivo, thereby accelerating wound healing and tissue regeneration.

[0021] Preferably, the scaffold is pre-seeded with cells related to dermal repair;

[0022] Preferably, the dermis repair-related cells include but are not limited to fibroblasts, mesenchymal stem cells, keratinocytes, vascular endothelial cells, other auxiliary cells or a combination thereof. Other auxiliary cells are selected from one or more of melanocytes, macrophages, and immunoregulatory cells.

[0023] Furthermore, the multi-layered skin dermal scaffold is characterized in that the scaffold is prepared by freeze drying, electrospinning, 3D printing, mold pressing or spraying.

[0024] The present invention also provides a method for preparing the multi-layered skin dermal scaffold, comprising the following steps:

[0025] (1) Modifying recombinant humanized type I collagen and recombinant humanized type III collagen to introduce cross-linking sites;

[0026] (2) preparing a collagen solution containing recombinant humanized type I collagen and recombinant humanized type III collagen;

[0027] (3) constructing a multilayered skin dermal scaffold layer by layer using the collagen solution by layer casting, layer-by-layer stacking, mold casting, electrospinning or 3D printing;

[0028] (4) curing and molding the constructed multi-layered skin dermal scaffold;

[0029] (5) sterilizing the solidified scaffold (e.g., sterilizing with gamma rays, ethylene oxide, or alcohol) to obtain the multi-layered skin dermal scaffold;

[0030] Preferably, one or more active ingredients that promote tissue repair are added to the collagen solution in step (2);

[0031] Preferably, the curing method in step (4) includes but is not limited to ultraviolet light irradiation, chemical cross-linking or freeze drying, preferably ultraviolet light irradiation or freeze drying;

[0032] Preferably, the functional component is introduced by immersion or adsorption after the curing and forming in step (4);

[0033] Preferably, cells related to dermal repair are pre-seeded in the multi-layered skin dermal scaffold in step (5).

[0034] The present invention also provides the use of the multi-layered skin dermal scaffold in preparing a biomaterial for skin repair, a skin repair-related drug delivery system, or a skin repair-related cell delivery system;

[0035] Preferably, the skin repair biomaterial is used for chronic wound repair, deep burn repair, full-thickness skin defect filling or other skin tissue engineering applications.

[0036] The multi-layered skin dermal scaffold provided by the present invention has good biocompatibility, mechanical support and tissue integration capabilities, and its application scenarios are wide, specifically including the following aspects:

[0037] 1. Deep burn wound repair: used for tissue reconstruction in areas with severe damage to the dermis after second-degree deep burns or third-degree burns, to promote angiogenesis and dermal remodeling, and improve wound healing quality.

[0038] 2. Repair of chronic refractory wounds: Suitable for chronic wounds such as diabetic foot, venous ulcers, and pressure sores. It serves as a tissue engineering scaffold to assist cell attachment and proliferation, thereby improving repair efficiency.

[0039] 3. Full-thickness skin defect substitute: used for full-thickness skin loss caused by trauma, surgical resection or deformity repair, as a dermis substitute in conjunction with epidermal transplantation to rebuild the complete skin structure.

[0040] 4. Cell therapy carrier: used as an attachment substrate for therapeutic cells such as fibroblasts and mesenchymal stem cells, and pre-cultured in vitro before transplantation to improve cell survival rate and repair function.

[0041] The beneficial effects of the present invention are:

[0042] The multi-layered skin dermal scaffold of the present invention is a three-layered skin dermal scaffold with recombinant humanized type I collagen and recombinant humanized type III collagen as its main components. This scaffold simulates the structural and functional differences between the papillary layer and the reticular layer in the natural dermis, and has good tissue adaptability and mechanical support properties. This makes it excellent in simulating dermal tissue structure, improving biological performance, enhancing cell integration, and promoting wound repair, while overcoming the limitations of traditional material sources and structural designs. Specifically:

[0043] 1. The scaffold structure of the present invention is close to the distribution of natural skin dermis, which can significantly improve the efficiency of cell directional attachment and tissue reconstruction;

[0044] 2. The use of recombinant humanized collagen avoids the risk of immune rejection and infection caused by animal-derived ingredients, and has higher biosafety;

[0045] 3. The multilayer structure allows for the regulation of mechanical properties, degradation rate, and cellular response to adapt to different wound environments;

[0046] 4. It can integrate active factors and multiple cells to improve the speed and effect of tissue repair;

[0047] 5. The scaffold preparation process is flexible and can be combined with 3D printing or mold remolding, making it suitable for large-scale production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic structural diagram of the multi-layered skin dermal scaffold based on recombinant humanized collagen of the present invention;

[0049] Figure 2 This is a general image of the recombinant humanized collagen dermal scaffold of Examples 1-3 of the present invention;

[0050] Figure 3 Microscope images and quantitative statistics of the scratch test in which the recombinant humanized collagen dermal scaffold promoted the migration of human fibroblasts in Examples 1-3 of the present invention;

[0051] Figure 4 These are confocal microscopy images of cells forming a network structure on the surface of the scaffold in Examples 1-3 of the present invention and Comparative Examples 1-2;

[0052] Figure 5 This is a general image of Example 3 of the present invention and the commercially available dermal stent Pinik promoting wound repair in a rabbit ear full-thickness skin defect animal model. DETAILED DESCRIPTION

[0053] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0054] Example 1

[0055] (1) 3D software design of multi-layered skin dermal scaffold based on recombinant humanized collagen

[0056] Based on the study of natural skin tissue structure, it was found that the hierarchical structure with dense middle and loose sides is more in line with the actual configuration of dermal tissue, among which the surface layer in contact with the wound surface presents a loose state with larger pores, which is conducive to early vascular ingrowth and rapid cell infiltration; the middle part of the scaffold maintains a higher density, providing the necessary mechanical support and structural stability. Accordingly, in the structural design stage, the present invention uses 3D modeling software to perform gradient simulation on the interlayer density of the scaffold to generate a pore structure parameter model that conforms to the "sparse outside-dense in the middle-sparse inside" pattern. The multi-layered skin dermal scaffold of the present invention is a three-layer structure, consisting of an upper material layer, a middle material layer and a lower material layer. The pore size of the upper material layer and the lower material layer is 1.5 to 2.1 mm (set to 1.9 mm in this embodiment), the pore size of the middle material layer is 400 to 1000 μm (set to 700 μm in this embodiment), and the porosity of the upper material layer, the middle material layer and the lower material layer is 60% to 70% (set to 69% in this embodiment). The thickness of each layer of the scaffold can be equal or unequal. The total thickness of the scaffold is determined according to the thickness of the animal's dermis, and the shape, size, and layer height of the scaffold are adjusted according to the part that needs to be repaired. The total thickness of the three layers of the scaffold determined according to the thickness of the rat's back dermis is 1.8 mm. The thickness of each layer can be equal (all three layers are 0.6 mm) or unequal (such as 300 μm for the upper layer, 600 μm for the middle layer, and 900 μm for the lower layer). In this embodiment, the thickness of each layer of the scaffold is 0.6 mm. If you encounter an animal with a thin dermis (such as rabbit ears), the thickness of the scaffold can be thinned. This design strategy can more accurately simulate the tissue characteristics of the natural skin dermis transitioning from the papillary layer to the reticular layer, thereby better structurally supporting cell attachment, angiogenesis, and tissue integration processes. The structural schematic diagram of the multi-layered skin dermis scaffold based on recombinant humanized collagen of the present invention is shown in FIG. Figure 1 shown.

[0057] (2) Synthesis of methacrylylated recombinant humanized collagen

[0058] Weigh 1g of recombinant humanized type I collagen and dissolve it in 50mL of deionized water. Once fully dissolved, adjust the solution to pH 7 and add 1mL of methacrylic anhydride dropwise. Allow to react at room temperature for 24 hours. Dialyze the solution in deionized water for 3 days using a 3500 molecular weight cutoff dialysis bag. Centrifuge at 10,000 rpm for 5 minutes to remove impurities. The supernatant was collected and lyophilized at -80°C to obtain methacryloylated recombinant humanized type I collagen (ColMA-I).

[0059] Weigh 1g of recombinant humanized type III collagen and dissolve it in 50mL of deionized water. Once fully dissolved, adjust the solution to pH 7 and add 1mL of methacrylic anhydride dropwise. Allow to react at room temperature for 24 hours. Dialyze the solution in deionized water for 3 days using a 3500 molecular weight cutoff dialysis bag. Centrifuge at 10,000 rpm for 5 minutes to remove impurities. The supernatant was collected and lyophilized at -80°C to obtain methacrylated recombinant humanized type III collagen (ColMA-III).

[0060] (3) Preparation of methacrylated recombinant humanized collagen solution

[0061] Take 20 mL of PBS and add it to a brown bottle containing 0.05 g of the initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 0.01 g of a water-soluble light absorber. Heat in a 45°C water bath to dissolve for 15 minutes, shaking several times. Take the required amount of methacrylated recombinant humanized type I collagen and methacrylated recombinant type III collagen at a 1:1 mass ratio and place them in a centrifuge tube. Add the required volume of the initiator standard solution to the tube and shake to fully soak the methacrylated recombinant humanized collagen. Heat in a 37°C water bath in the dark for 5 minutes, shaking several times. Centrifuge at 3000 rpm for 2 minutes to remove any bubbles until the solution is completely homogenized and remains light yellow and clear. This yields a 10% (w / v) methacrylated recombinant humanized collagen solution. Finally, 2% (w / w) polyethylene glycol diacrylate (PEGDA) was added to increase the printability of the solution.

[0062] (4) High-resolution DLP printing equipment and parameter settings

[0063] Using a prepared methacryloyl-modified recombinant humanized collagen solution as the printing ink, a high-precision projection-based light-curing bio-3D printer, the BP8601Pro, was used to prepare a three-layer skin dermal scaffold composed primarily of recombinant humanized collagen. The scaffolds were printed with an exposure intensity of 13, an exposure time of 10 seconds, and a slice height of 100 μm. After printing, the deposition platform was slowly removed, and the printed part was scraped off the bottom with a coverslip. The printed part was then immersed in a PBS solution for 5 minutes for development.

[0064] (5) Sterilization and preservation of recombinant humanized collagen three-layer structure skin dermal scaffold

[0065] Soak the printed scaffold in a 75% ethanol solution for 15 minutes for preliminary sterilization, then rinse twice with sterile PBS buffer for 10 minutes each time to remove residual alcohol. Remove the cleaned scaffold and place it in a freezing container with sterile tweezers. Pre-freeze it at -80°C for at least 4 hours, and then place it in a freeze dryer for vacuum freeze-drying to obtain a dry and stable scaffold sample. If not used in the short term, the freeze-dried scaffold should be sealed and stored at -80°C to maintain its structural and functional integrity. Before use, remove the scaffold and sterilize it under ultraviolet light for 30 minutes to further reduce the risk of microbial contamination and ensure its safe use in cell experiments or in vivo transplantation.

[0066] Example 2

[0067] The difference between this embodiment and embodiment 1 is that in step (3), the required mass of methacryloyl-recombinant humanized type I collagen and methacryloyl-recombinant humanized type III collagen are taken to make the mass ratio of the two be 3:2. The other steps are the same as those in embodiment 1.

[0068] Example 3

[0069] The difference between this embodiment and embodiment 1 is that in step (3), the required mass of methacryloyl-recombinant humanized type I collagen and methacryloyl-recombinant humanized type III collagen are taken to make the mass ratio of the two be 4:1. The other steps are the same as those in embodiment 1.

[0070] The general image of the recombinant humanized collagen dermal scaffold invented in Examples 1-3 of the present invention is as follows Figure 2 shown.

[0071] Comparative Example 1

[0072] The difference between Comparative Example 1 and Example 1 is that 1% (w / w) fibronectin (FN) is added in step (3). After uniform mixing, the liquid becomes a yellow suspension, which does not affect the printability of the liquid. The other steps are the same as in Example 1.

[0073] Comparative Example 2

[0074] The difference between Comparative Example 1 and Example 1 is that 5% (w / w) fibronectin (FN) is added in step (3). After uniform mixing, the liquid becomes a yellow suspension, which does not affect the printability of the liquid. The other steps are the same as in Example 1.

[0075] Experimental Example 1

[0076] To evaluate the effects of different treatment groups on the migration ability of fibroblasts (FBs), a scratch test was used for observation and quantitative analysis. FBs were seeded in 6-well plates, with approximately 2×10 5 Cells were cultured until the monolayer was confluent (about 90-100% confluence). Use a 1000μL pipette tip to draw a straight line vertically in the middle of the cell monolayer to form a wound. Use the same force and direction for each well. Gently rinse with PBS 2-3 times to remove floating cells and debris. Replace with serum-free culture medium, and add treatment solution (the treatment solution is the extract of the scaffold soaked in culture medium) of different scaffold groups (such as 1:1, 3:2, 4:1 ratios). The control group did not receive any treatment. Take a picture at 0h to record the initial width of the scratch, and then take pictures again at 24h and 48h. Use ImageJ software to measure the change in scratch area or width, and calculate the wound closure rate: Wound Closure (%) = W0-Wt / Wt×100%, where W0 is the scratch width at 0h, and Wt is the scratch width at each time point. Use t-test or one-way analysis of variance (ANOVA) to evaluate the significance between different groups, and p<0.05 is considered statistically significant. As Figure 3 By comparing Examples 1, 2 and 3 with the control group, it can be seen that all Examples significantly promoted wound closure at 24h and 48h (p < 0.01 or p < 0.001). Among them, Example 2 showed the highest wound healing rate at both time points, and the wound closure at 24h was significantly faster than that of Example 1 and Example 3, suggesting that it has the strongest promoting effect on cell migration. At 48h, the healing effect of Example 3 group was close to that of Example 2 group, but slightly lower than the latter, while Example 1 group was relatively weakest. The overall trend shows that the appropriate ratio can significantly enhance the migration ability of fibroblasts, which is beneficial to the tissue repair process.

[0077] Experimental Example 2

[0078] To observe the effects of different material ratios and fibronectin (FN) addition on the adhesion and cytoskeleton structure of endothelial cells (ECs) and fibroblasts (FBs), CellMask TM Green Plasma Membrane Stain (Thermo Fisher) was used for cytoskeleton staining. ECs and FBs were seeded on the pretreated material surface (i.e., the scaffold surface. If the sample was not freeze-dried, the pretreatment was disinfection; if the sample was freeze-dried, the pretreatment was soaking in culture medium overnight). The cell seeding density was 2×10 4 cells / cm 2, and then stained after 24 hours of culture. The cells were fixed with 4% paraformaldehyde (PFA) for 10-15 minutes, washed 3 times with PBS, and incubated with CellMask dye (1:1000 dilution) for 30 minutes at room temperature in the dark. Then they were washed with PBS again, and the nuclei were stained with DAPI (1μg / mL) for 10 minutes. Finally, images were collected under a fluorescence confocal microscope to observe the green fluorescent labeled cytoskeleton and the blue DAPI labeled cell nucleus respectively. The results are shown in Figure 2. Figure 4 shown.

[0079] In endothelial cells (ECs), all treatment groups showed a certain degree of adhesion and cytoskeletal spreading. Among them, Example 3 had a higher cell density and sufficient spreading, showing good cell adhesion ability. In contrast, although the cell morphology of Examples 1 and 2 groups was clear and well spread, the cell number was slightly lower. It is worth noting that after adding fibronectin (1% or 5% FN) to the Example 3 group, the cytoskeletal structure was significantly improved, especially in the 5% FN treatment group, where the cells were more evenly distributed and spread radially, indicating that FN can significantly promote the adhesion and morphological maintenance of ECs.

[0080] Fibroblasts (FBs) exhibited well-defined cytoskeleton structures in all treatment groups, demonstrating strong adaptability. The FN-added group also showed further expansion of cell morphology, suggesting that high concentrations of FN also have a certain promoting effect on FBs, but the degree of effect is weaker than that on ECs.

[0081] In summary, the material ratio has a regulatory effect on cell adhesion behavior, and the addition of fibronectin is particularly helpful in improving cell spreading and skeleton formation under adverse conditions.

[0082] Experimental Example 3

[0083] To evaluate the efficacy of a collagen scaffold in deep skin wound repair, a rabbit ear skin defect model was constructed. Healthy adult rabbits (weighing 2.0-2.5 kg) were anesthetized with an intramuscular injection of the compound anesthetic Sumianxin before the experiment. After the animals were immobilized, a 1-cm-diameter circular biopsy instrument was used to vertically excise the epidermis and dermis from the inner side of the rabbit ear until the perichondrium was exposed, creating a standardized full-thickness skin defect.

[0084] Multiple wounds were established on each rabbit ear and the rabbits were randomly divided into the following three groups for treatment:

[0085] In the blank control group, the wound surface was left naturally exposed without any material intervention. In the collagen scaffold group, a pre-prepared collagen scaffold was applied to the wound surface, and a breathable but water-repellent medical PU film was applied to the wound surface to maintain local moisture and secure the scaffold. In the medical dressing group, the wound surface was covered with the commercially available medical biological dressing Pelnac, serving as a positive control. Wound photographs were taken on days 2, 7, and 14 postoperatively, and the healing process was recorded. Evaluation criteria included wound color change, shrinkage area, and the growth of scab and granulation tissue.

[0086] like Figure 5 As shown, the wounds of each group were observed with the naked eye on the 2nd, 7th and 14th days after surgery and the healing progress was recorded:

[0087] Day 2 (Acute Inflammation Phase): The blank group experienced significant wound redness and swelling, accompanied by exudate and bleeding; the Example 3 group experienced moist wounds with regular edges, and the local inflammatory response was reduced; the Pelnac group, covered by a dressing, had a rosy, but no significant exudate, indicating a milder initial reaction. Day 7 (Granulation Tissue Proliferation Phase): In the blank group, scabs or necrotic tissue were visible in the center of the wound, with limited granulation at the edges; in the Example 3 group, dense granulation tissue had formed, the wound surface was significantly reduced, and new epithelial growth was visible at the edges; the Pelnac group had a relatively complete wound surface, but the granulation tissue was slightly sparse. Day 14 (Tissue Reconstruction Phase): Parts of the blank group's wounds remained partially closed, with significant localized pigmentation and poor healing quality; the collagen scaffold group had essentially closed, with a rosy, smooth surface, indicating a better repair effect; while the Pelnac group experienced significant wound contraction, slight depression was still visible in the central area, indicating a slightly inferior healing process to that of the Example 3 group.

[0088] Comprehensive observation results show that Example 3 of the present invention combined with the application of PU film can significantly promote the tissue repair of full-thickness skin defects in rabbit ears, has good wound environment regulation ability and tissue regeneration promotion effect, and is better than the blank and commercially available medical dressing groups.

[0089] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A multi-layered skin dermal scaffold, characterized in that: The multi-layer skin dermal scaffold has a three-layer structure, consisting of an upper material layer, a middle material layer and a lower material layer. The upper material layer, the middle material layer and the lower material layer are composed of recombinant type I humanized collagen and recombinant type III humanized collagen. The pore size of the upper material layer and the lower material layer is 1.5 to 2.1 mm, the pore size of the middle material layer is 400 to 1000 μm, and the porosity of the upper material layer, the middle material layer and the lower material layer is 60% to 70%.

2. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The mass ratio of the recombinant humanized type I collagen to the recombinant humanized type III collagen is 1:1 to 4:1; Preferably, the mass ratio of the recombinant humanized type I collagen to the recombinant humanized type III collagen is 3:

2.

3. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The cross-linking degree of the multi-layered skin dermal scaffold is 40% to 70%.

4. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The pore size of the upper material layer and the lower material layer is 1.9 mm, and the pore size of the middle material layer is 700 μm.

5. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The thickness of the multi-layered skin dermal scaffold is determined by the thickness of the animal dermis layer, and the thicknesses of the upper material layer, the middle material layer and the lower material layer are equal or unequal.

6. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The recombinant humanized type I collagen and the recombinant humanized type III collagen are modified by one or more chemical or physical methods; Preferably, the modification methods include but are not limited to methacrylation, EDC / NHS cross-linking, photosensitive cross-linking, enzyme cross-linking or functional group grafting.

7. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The scaffold further comprises one or more active ingredients that promote tissue repair; Preferably, the active ingredients that promote tissue repair include but are not limited to VEGF, bFGF, aFGF, fibronectin, anti-inflammatory drugs, antimicrobial peptides, anti-infective ingredients or small molecule regeneration regulators; Preferably, the active ingredient that promotes tissue repair is loaded into the scaffold by physical adsorption, blending, cross-linking or slow-release microcarriers; Preferably, the sustained-release microcarrier comprises microspheres, nanoparticles, liposomes or hydrogel particles; Preferably, the scaffold further comprises a functional component; Preferably, the functional components include but are not limited to pro-angiogenic factors, plasmid DNA, mRNA or siRNA expressing specific growth factors; Preferably, the scaffold is pre-seeded with cells related to dermal repair; Preferably, the dermis repair-related cells include but are not limited to fibroblasts, mesenchymal stem cells, keratinocytes, vascular endothelial cells, other auxiliary cells or a combination thereof, and the other auxiliary cells are selected from one or more of melanocytes, macrophages, and immunoregulatory cells.

8. The multi-layered skin dermal scaffold according to claim 1, characterized in that: The scaffold is prepared by freeze drying, electrostatic spinning, 3D printing, mold pressing or spraying.

9. A method for preparing the multi-layered skin dermal scaffold according to claim 1, characterized in that: The following steps are involved: (1) Modifying recombinant humanized type I collagen and recombinant humanized type III collagen to introduce cross-linking sites; (2) preparing a collagen solution containing recombinant humanized type I collagen and recombinant humanized type III collagen; (3) constructing a multilayered skin dermal scaffold layer by layer using the collagen solution by layer casting, layer-by-layer stacking, mold casting, electrospinning or 3D printing; (4) curing and molding the constructed multi-layered skin dermal scaffold; (5) sterilizing the solidified scaffold to obtain the multi-layered skin dermal scaffold; Preferably, one or more active ingredients that promote tissue repair are added to the collagen solution in step (2); Preferably, the curing method in step (4) includes but is not limited to ultraviolet light irradiation, chemical cross-linking or freeze drying, preferably ultraviolet light irradiation or freeze drying; Preferably, the functional component is introduced by immersion or adsorption after the curing and forming in step (4); Preferably, cells related to dermal repair are pre-seeded in the multi-layered skin dermal scaffold in step (5).

10. Use of the multilayered skin dermal scaffold according to claim 1 in the preparation of a biomaterial for skin repair, a drug delivery system related to skin repair, or a cell delivery system related to skin repair; Preferably, the skin repair biomaterial is used for chronic wound repair, deep burn repair, full-thickness skin defect filling or other skin tissue engineering applications.

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