A new composite collagen material for wound repair

Abstract

The present invention relates to the field of biomedical materials, particularly to a novel composite collagen material for wound repair, and more specifically to a method for preparing a novel composite collagen material comprising recombinant humanized collagen type 21 (ColpepA1 21) and its use in wound repair. The present invention utilizes ColpepA1 21 to significantly promote key biological reactions in the wound healing process, including accelerating cell migration, enhancing skin hydration and barrier function, and improving the stability and elasticity of skin structure. The resulting novel composite collagen material effectively promotes wound healing and cell regeneration, alleviating oxidative damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a preparation method of a novel composite collagen material for wound repair and its application in wound repair, including a preparation method and application of a novel composite collagen material composed of type 21 recombinant humanized collagen and a composition thereof. Background Art

[0002] Collagen is not only one of the most abundant proteins in the human body, accounting for over 30% of the total protein content, but it is also the cornerstone for maintaining the structure and function of various tissues. It is present in the skin, bones, tendons, blood vessels, and other connective tissues, forming a three-dimensional network that provides mechanical strength and stability. In the skin, collagen is responsible for maintaining skin firmness and elasticity, and maintaining a smooth and radiant surface. With aging, the body's natural production of collagen slows, and existing collagen is gradually degraded and damaged by factors such as UV exposure, environmental pollution, and unhealthy lifestyle habits. This causes the skin to gradually lose its original elasticity and firmness, resulting in signs of aging such as sagging, fine lines, and wrinkles, as well as decreased wound healing ability. Furthermore, the reduction of collagen in other tissues can affect bone density, tendon elasticity, and vascular stability, thereby impacting overall health. In the field of wound repair, traditional treatments face challenges in accelerating wound healing, reducing scar formation, and improving repair quality. Although there are many products on the market for promoting wound healing, such as collagen-based dressings and growth factors, how to effectively promote wound healing, improve treatment efficacy and alleviate patient pain remains an important research direction.

[0003] In this context, in-depth research on specific collagen types, such as type 21 collagen (COL21A1), is particularly important. Type 21 collagen is a non-fibrillar collagen that is relatively rare in the adult human body, accounting for less than 1% of the total collagen composition. Compared to other, more well-known collagen types, type 21 collagen has been relatively understudied. Type 21 collagen belongs to the small molecule collagen family. As part of the FACIT (fibril-associated collagen with interrupted triple helices) family, type 21 collagen plays a crucial role in connecting extracellular matrix components. According to research by Chou MY and Li HC (March 2002), type 21 collagen is primarily expressed in vascular smooth muscle cells and may play a role in extracellular matrix assembly during angiogenesis. Furthermore, the gene expression of type 21 collagen is regulated by developmental stage, with higher expression levels during fetal development, suggesting that it may play an important role in the development of various tissues. In recent years, research on type 21 collagen has revealed its potential role in vascular health and blood pressure regulation. Surendran et al. (2016) conducted a large-scale genotyping survey and found that the type 21 collagen gene is involved in collagen formation in various tissues, including the heart and aorta, and is associated with vascular remodeling and hypertension. This provides new insights into the role of COL21A1 in promoting skin health and anti-aging. These properties give type 21 collagen unique potential for applications in skin health, anti-aging, and vascular health.

[0004] Although research on the effects of type 21 collagen on vascular health and blood pressure regulation has been conducted, its application in promoting wound repair is still in the exploratory stage. The development of new composite materials based on type 21 collagen can not only provide more effective treatment options for wound repair, but also improve the patient's recovery quality and quality of life by promoting high-quality tissue regeneration. Therefore, the present invention aims to develop a new type of composite collagen material and its preparation method, and to provide a more effective, safe and biocompatible treatment plan for wound repair through in-depth research on the biological properties of type 21 collagen and its mechanism of action in wound repair. In addition, the present invention also explores the synergistic effect of type 21 collagen with other bioactive molecules to achieve precise regulation of the wound healing process, thereby accelerating wound healing, reducing scar formation, and improving the quality of repaired tissue. Summary of the Invention

[0005] The invention relates to a preparation method of a novel composite collagen material and application of the collagen material in wound repair.

[0006] In one aspect, the present invention provides a method for preparing a collagen material for wound repair, wherein the method comprises the following steps: (1) mixing collagen with auxiliary components; (2) solidifying and shaping the collagen material; and (3) sterilizing the collagen material; wherein the collagen material comprises type 21 recombinant humanized collagen, and the amino acid sequence of the collagen comprises the amino acid sequence shown in SEQ ID No. 1:

[0007] GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGL PGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG.

[0008] In one embodiment, the mixing of the present invention is performed at room temperature or under heating.

[0009] In one embodiment, the mixing of the present invention is carried out at room temperature of 20-25°C; preferably, the mixing is carried out at any one or more temperatures of 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C; preferably, the mixing is carried out at 25°C.

[0010] In one embodiment, the solidification molding of the present invention is carried out at room temperature or low temperature.

[0011] In one embodiment, the coagulation molding of the present invention refers to being carried out at a low temperature of 2-15°C; preferably, the coagulation molding is carried out at any one or more temperatures of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, and 15°C.

[0012] In one embodiment, the coagulation molding of the present invention is carried out at room temperature of 20-25°C; preferably, the coagulation molding is carried out at any one or more temperatures of 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C; preferably, the coagulation molding is carried out at 25°C.

[0013] In one embodiment, the sterilization of the present invention comprises any one of heat sterilization, chemical sterilization or radiation sterilization.

[0014] Preferably, the sterilization is gamma ray sterilization or plasma sterilization.

[0015] In one embodiment, the method of the present invention comprises the following steps: cross-linking a mixed material prepared by proportioning collagen and auxiliary ingredients in a sodium acetate buffer at room temperature; transferring the mixed solution into a mold, allowing it to stand, and refrigerating it for 12-24 hours to form the mold; and performing low-temperature curing and plasma sterilization.

[0016] In one embodiment, the method of the present invention comprises the following steps: pre-soaking collagen and auxiliary components in water and heating to dissolve; adding hyaluronic acid solution, stirring and solidifying at room temperature; low-temperature solidification, and plasma sterilization.

[0017] In one embodiment, the method of the present invention comprises the following steps: mixing collagen and auxiliary components, spin coating on a substrate; drying and peeling, and sterilizing with gamma rays.

[0018] In one embodiment, the method of the present invention comprises the following steps: mixing collagen and auxiliary ingredients, pouring into a mold, freezing, and then freeze-drying; gas cross-linking, and gamma ray sterilization.

[0019] In one embodiment, the auxiliary ingredients of the present invention are selected from one or more of the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, and the like.

[0020] In one embodiment, the concentration of the collagen is 0.0001% to 50% based on the total weight of the collagen material.

[0021] Preferably, the concentration of the collagen is 1% to 20%.

[0022] In one embodiment, the weight ratio of the collagen and auxiliary ingredients of the present invention is selected from:

[0023] Collagen and antimicrobial agent: 5:1 to 20:1; or

[0024] Collagen and anti-inflammatory agent: 20:1 to 100:1; or

[0025] Collagen to growth factor: 50:1 to 200:1; or

[0026] Collagen and moisturizer: 1:1 to 10:1; or

[0027] Collagen to antioxidants: 10:1 to 50:1; or

[0028] Collagen and emulsifier or thickener: one or more combinations in a ratio of 5:1 to 20:1.

[0029] In one embodiment, the method of the present invention comprises the following steps: cross-linking a mixed material composed of type 21 collagen, hyaluronic acid, and a cross-linking agent in a sodium acetate buffer at room temperature; transferring the mixed solution into a mold, allowing it to stand, and refrigerating it for 12-24 hours to form; and low-temperature curing and plasma sterilization.

[0030] Preferably, the ratio is: 2-3% (w / v) type 21 collagen, 1% (w / v) hyaluronic acid, 0.1% (w / v) cross-linking agent; more preferably, the ratio of type 21 collagen is 3% (w / v).

[0031] Preferably, the cross-linking agent is EDC.

[0032] In one embodiment, the method of the present invention comprises the following steps: pre-soaking a mixed material of gelatin and collagen in water and heating to dissolve; adding a hyaluronic acid solution, stirring and solidifying at room temperature; curing at low temperature, and plasma sterilization.

[0033] Preferably, the ratio is: 2% (w / v) type 21 collagen solution, 1% (w / v) hyaluronic acid, and 3% (w / v) gelatin.

[0034] In one embodiment, the method of the present invention comprises the following steps: mixing type 21 collagen and PLGA in a certain proportion, and spin-coating the mixture on a substrate; drying and peeling the mixture, and sterilizing the mixture with gamma rays.

[0035] Preferably, the ratio is: 1.5% (w / v) type 21 collagen solution, 2% (w / v) PLGA.

[0036] In one embodiment, the method of the present invention comprises the following steps: mixing type 21 collagen and chitosan in a certain proportion, pouring the mixture into a mold, freezing the mixture, and then freeze-drying the mixture; gas cross-linking, and gamma ray sterilization.

[0037] Preferably, the ratio is: 2.5% (w / v) type 21 collagen solution, 1.5% (w / v) chitosan.

[0038] In one aspect, the present invention provides a type 21 recombinant humanized collagen, wherein the amino acid sequence of the collagen is selected from SEQ ID No. 1.

[0039] In one embodiment, the collagen of the present invention has the activity of promoting tissue repair and regeneration, especially accelerating the wound healing and cell remodeling process after skin damage.

[0040] In one embodiment, the collagen of the present invention has the activity of promoting cell migration, thereby helping to shorten wound healing time and improve the self-recovery ability of the skin.

[0041] In one embodiment, the collagen of the present invention can enhance skin moisture regulation and optimize barrier function.

[0042] In one embodiment, the collagen of the present invention can promote the expression of the water channel protein AQP3, thereby maintaining the appropriate hydration state of the wound surface and the surrounding skin by improving the skin's water retention capacity and reducing the water evaporation rate, thereby promoting the wound repair process.

[0043] In one embodiment, the collagen of the present invention can enhance the stability and elasticity of the skin structure, thereby playing a key role in the repair and regeneration of damaged skin.

[0044] In one embodiment, the collagen of the present invention can promote the formation and maintenance of the skin barrier, thereby playing a key role in the repair and regeneration of damaged skin.

[0045] In one embodiment, the collagen of the present invention can enhance the expression of FLG;

[0046] In one embodiment, the collagen of the present invention can enhance the expression of LOR;

[0047] In one embodiment, the collagen of the present invention can increase the activity of TGM1.

[0048] In one embodiment, the collagen of the present invention can be used in combination with moisturizers such as hyaluronic acid and glycerin.

[0049] In one embodiment, the collagen of the present invention can be combined with antioxidants such as vitamin E, green tea extract, etc. to enhance the skin's ability to resist environmental oxidative damage.

[0050] In one embodiment, the collagen of the present invention may further be added with a stabilizer such as vitamin B3 to maintain the stability and activity of the collagen.

[0051] In one embodiment, the collagen of the present invention may also be used in combination with an emulsifier to stabilize the emulsification system of the product and ensure uniform distribution of the active ingredients.

[0052] In one embodiment, the collagen of the present invention is combined with a penetration enhancer, such as liposomes or nanoparticles, to enhance the permeability of the collagen in the skin.

[0053] In one embodiment, the collagen of the present invention can be used in combination with excipients and fillers to improve the texture and appearance of the product.

[0054] In one embodiment, the moisturizing ingredients may include hyaluronic acid, glycerin, natural moisturizing factor (NMF), allantoin, polyols (such as butylene glycol, glycol, etc.), plant extracts (such as aloe extract, green tea extract, etc.); the antioxidant ingredients may include vitamin C and its derivatives, vitamin E and its derivatives, conjugated linoleic acid, white tea extract, green tea extract, resveratrol, glutathione, etc.; the whitening ingredients may include arbutin, kojic acid, vitamin B3 (niacinamide), glycyrrhizic acid, fruit acids (such as citric acid, lactic acid, etc.) and other herbal extracts.

[0055] Another aspect of the present invention provides a novel composite collagen material prepared according to the above method, wherein the collagen material is used for repairing wounds after skin damage.

[0056] On the other hand, the present invention provides a novel composite collagen material for wound repair, wherein the novel composite collagen material comprises type 21 recombinant humanized collagen, the amino acid sequence of the collagen is selected from SEQ ID No. 1, and further comprises one or more auxiliary ingredients selected from the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, etc.

[0057] In one embodiment, the novel composite collagen material of the present invention has a collagen concentration of 0.0001% to 50% based on the total weight of the collagen material.

[0058] Preferably, the concentration of the collagen is 1% to 20%.

[0059] In one embodiment, the novel composite collagen material of the present invention is designed for wound repair, and uses the collagen as an active ingredient to accelerate the wound healing process, reduce scar formation, and improve the treatment effect.

[0060] In one embodiment, the collagen material of the present invention, wherein the weight ratio of the collagen to the auxiliary components is selected from one or more of the following combinations:

[0061] In one embodiment, the ratio of collagen to antimicrobial agent is selected from 5:1 to 20:1; an appropriate concentration of antimicrobial agent can effectively inhibit bacterial growth, but too high a concentration may cause skin irritation.

[0062] In one embodiment, the ratio of collagen to anti-inflammatory agent is selected from 20:1 to 100:1; the use of anti-inflammatory agent should be carefully controlled to avoid causing local or systemic side effects.

[0063] In one embodiment, the ratio of collagen to growth factor is selected from 50:1 to 200:1; growth factors are highly active and can be effective in small doses, while excessive amounts may accelerate tumor cell growth or other adverse reactions.

[0064] In one embodiment, the ratio of collagen to moisturizer is selected from 1:1 to 10:1; a higher amount of moisturizer can enhance the moisturizing property of the skin, but it should not be excessive so as not to affect other functions of the formula.

[0065] In one embodiment, the ratio of collagen to antioxidant is selected from 10:1 to 50:1; the antioxidant can prevent the preparation from oxidation, but its dosage should be controlled so as not to affect the long-term stability of the product.

[0066] In one embodiment, the ratio of collagen to emulsifier or thickener is selected from 5:1 to 20:1; it helps to improve the texture and stability of the product, but should be used in moderation to ensure the applicability and comfort of the final product.

[0067] In one embodiment, the novel composite collagen material of the present invention is suitable for repairing wounds after various types of skin injuries.

[0068] In one embodiment, the skin injury includes, but is not limited to, a surgical incision, a burn, an abrasion, or a chronic wound.

[0069] The comparative raw material of the present invention can effectively accelerate the healing process and reduce scar formation.

[0070] In one embodiment, the novel composite collagen material of the present invention may be in the form of, but not limited to, solid, liquid, semisolid or gel preparations.

[0071] In one embodiment, the novel composite collagen material of the present invention may be in the form of a smearable gel, cream, dressing, spray, oral / internal medication, or injection.

[0072] In one embodiment, the novel composite collagen material of the present invention is a collagen-hyaluronic acid hydrogel dressing, which is prepared by a chemical cross-linking method:

[0073] Material composition: Type 21 collagen: 2-3% (w / v), hyaluronic acid: 1% (w / v), EDC: 0.1% (w / v) (cross-linking agent);

[0074] Preparation process:

[0075] The mixed materials were cross-linked in sodium acetate buffer at room temperature;

[0076] Transfer the mixture into a mold, let it stand, and then refrigerate for 12-24 hours to completely gel;

[0077] Low temperature plasma sterilization, sterile packaging.

[0078] In one embodiment, the novel composite collagen material of the present invention is a collagen-hyaluronic acid hydrogel dressing, and gelatin is used as a cross-linking agent to prepare the collagen-hyaluronic acid hydrogel dressing:

[0079] Material composition: Type 21 collagen solution: 2% (w / v), hyaluronic acid: 1% (w / v), gelatin: 3% (w / v);

[0080] Preparation process:

[0081] Gelatin and collagen are pre-soaked in water and heated to dissolve;

[0082] Add hyaluronic acid solution, stir and solidify at room temperature;

[0083] Low temperature curing, plasma sterilization.

[0084] In one embodiment, the novel composite collagen material of the present invention is a film dressing, which is prepared by the following method:

[0085] Material composition: Type 21 collagen solution: 1.5% (w / v), PLGA: 2% (w / v);

[0086] Preparation process:

[0087] Collagen and PLGA were mixed and spin-coated on the substrate;

[0088] Dry and peel, gamma sterilize.

[0089] In one embodiment, the novel composite collagen material of the present invention is a sponge or fiber dressing, which is prepared by the following method:

[0090] Material composition: Type 21 collagen solution: 2.5% (w / v), chitosan: 1.5% (w / v);

[0091] Preparation process:

[0092] Mix collagen and chitosan, pour into molds, freeze and then freeze-dry;

[0093] Gas cross-linking and gamma ray sterilization.

[0094] In one embodiment, the novel composite collagen material of the present invention promotes rapid and high-quality wound repair, optimizes the healing process, reduces inflammatory response, accelerates skin regeneration, reduces scar formation, and improves treatment efficacy by providing ColpepA1 21.

[0095] Another aspect of the present invention provides a use of type 21 recombinant humanized collagen in the preparation of a medical composition for wound repair, wherein the amino acid sequence of the collagen comprises the amino acid sequence shown in SEQ ID No.1.

[0096] In one embodiment, the composition of the present invention is a solid, liquid, semisolid or gel formulation.

[0097] In one embodiment, the composition of the present invention is in the form of a formulation selected from one or more of a smearable gel, a cream, a dressing, a spray, an oral medication, or an injection.

[0098] In one embodiment, the composition of the present invention is in the form of a dressing, including but not limited to a hydrogel dressing, a film dressing, a sponge or a fiber dressing.

[0099] In one embodiment, the composition of the present invention is in the form of a hydrogel dressing, more preferably a collagen-hyaluronic acid hydrogel dressing.

[0100] Another aspect of the present invention provides a use of any of the aforementioned collagen materials in the preparation of a medical composition for wound repair.

[0101] The collagen and its composition of the present invention or the novel composite collagen material prepared therefrom have the following characteristics:

[0102] 1. Rarity and Unique Applications: Type 21 collagen is relatively rare in the adult human body and has been relatively understudied. Furthermore, research indicates that its potential applications in skin and vascular health are unique. The development of wound repair materials using this rare collagen type demonstrates the innovative nature of exploring new biomaterials.

[0103] 2. Promote natural skin healing and HaCaT cell migration: Type 21 collagen accelerates the skin's natural healing process by promoting HaCaT cell migration, which is crucial for wound repair. Accelerating the formation and healing of new skin based on HaCaT cell migration is the key to this innovative application.

[0104] 3. Ability to enhance AQP3 expression: AQP3 is an important water channel protein that is critical for skin hydration and cell migration. Type 21 collagen can enhance AQP3 expression, which not only helps retain moisture in wounds but also may promote more efficient cell migration and wound healing.

[0105] Advantages of the present invention

[0106] 1. Promote rapid healing: By promoting HaCaT cell migration and enhancing AQP3 expression, type 21 collagen can accelerate the wound healing process and shorten the patient's recovery time.

[0107] 2. Improve repair quality: Promote the production of type I and type III collagen, help build new skin tissue with stable structure and normal function, and improve repair quality.

[0108] 3. Biocompatibility and low immunogenicity: As a protein naturally present in the human body, type 21 collagen has good biocompatibility and low immunogenicity, reducing the risk of triggering an immune response.

[0109] 4. Multifunctionality: In addition to directly promoting wound healing, type 21 collagen may also improve the skin's water retention capacity through its effect on AQP3 expression, and has potential added value in improving skin health. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 The figure shows the healing process of keratinocytes in a scratch test, including the closure of the scratch at different time points.

[0111] Figure 2 and Figure 3 The figure shows a comparison of the healing rates of keratinocyte scratch wounds, demonstrating the quantitative analysis results of the healing progress of different experimental tissues.

[0112] Figure 4 The figures show the aquaporin 3 content in cells after different treatments, and the effect of ColpepA1 21 on AQP3 expression is shown by comparison.

[0113] Figure 5 Relative integrated optical density (IOD) values ​​of filaggrin (FLG) are shown.

[0114] T-test was used to test the significant differences between the corresponding experimental groups and the negative control group, where significance is indicated by *, * indicates p < 0.05, and ** indicates p < 0.01.

[0115] Figure 6 Relative integrated optical density (IOD) values ​​of loricrin (LOR) are shown.

[0116] Figure 7 Relative integrated optical density (IOD) values ​​of transglutaminase 1 (TGM1) are shown.

[0117] Figure 8 Shown is the effect of ColpepA1 21 hydrogel dressing on cell activity under oxidative stress environment.

[0118] Related definitions

[0119] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0120] As used herein, the term "collagen (COL)" refers to a vital structural protein in the human body, found in a variety of tissues and organs, such as skin, bones, tendons, ligaments, and cornea. The primary function of these proteins is to provide strength, stability, and elasticity to body tissues, ensuring the normal functioning of various physiological functions. Currently, there are at least 28 known types of collagen, each with a unique role and function in the body, forming a complex and finely regulated biological system.

[0121] As used herein, the term "type 21 collagen (COL21A1)" refers to a type of collagen found in the human body that belongs to the small molecule collagen family. As part of the FACIT (fibril-associated collagen with interrupted triple helix) family, type 21 collagen plays a vital role in connecting components of the extracellular matrix. Through its unique molecular structure, this collagen promotes interactions and linkages between different collagen types, thereby supporting the integrity and order of the extracellular matrix and its function in wound healing and tissue repair. Although it is not as well-known as type I and type III collagen, it plays a role in specific tissues and physiological processes, involving the composition of the extracellular matrix and skin repair mechanisms. Although type 21 collagen is relatively rare in the adult human body, accounting for less than 1% of the total collagen, its possible role in cellular processes such as migration, proliferation, and differentiation, as well as its potential role in maintaining extracellular matrix structure and promoting tissue regeneration, are important directions for future research.

[0122] As used herein, the term "recombinant humanized collagen type 21 (ColpepA1 21)" refers to a protein derived through genetic recombination technology that mimics the type 21 collagen naturally produced by the human body. This protein, encoded by a specific amino acid sequence, is designed to promote wound healing, support new tissue formation, and enhance the efficiency of the skin repair process.

[0123] As used herein, the term "wound repair / wound healing" refers to a series of complex biological processes initiated by the skin after injury, including inflammatory response, formation of new tissue, proliferation and migration of keratinocytes and fibroblasts, and deposition and reconstruction of new extracellular matrix, aiming to restore the integrity and function of the skin.

[0124] As used herein, the term "HaCaT (human keratinocytes)" refers to an immortalized human keratinocyte cell line that is widely used to study skin biology, skin pathology, and drug toxicity testing. It is considered a valuable research tool because it can mimic the behavior of normal epidermal cells.

[0125] As used herein, the term "HFF-1 (human fibroblasts)" refers to fibroblasts isolated from the forearm of human fetuses. They are an important component of skin structure, responsible for synthesizing collagen and other extracellular matrix proteins, and are often used to study skin aging, wound healing, and collagen synthesis.

[0126] As used herein, the term "cell migration" refers to the process by which cells move from one location to another in an in vivo or in vitro environment, which is an important biological process in skin wound healing and tissue regeneration.

[0127] As used herein, the term "aquaporin AQP3" refers to an aquaporin protein that is primarily expressed in skin cells and is responsible for regulating cellular water transport, playing a key role in maintaining skin moisture balance and barrier function.

[0128] As used herein, the terms "type I collagen" and "type III collagen" refer to the skin's primary structural proteins, responsible for maintaining its strength, elasticity, and firmness, respectively. Type I collagen is the most abundant type of collagen and is primarily found in skin, bone, and connective tissue. Type III collagen, on the other hand, is primarily found in the extracellular matrix of the skin and plays a crucial role in skin elasticity and repair processes.

[0129] As used herein, the term "TGFβ1" (transforming growth factor β1) refers to a multifunctional cytokine that regulates cell proliferation, differentiation, and migration. In skin physiology, TGFβ1 has an important influence on collagen synthesis, wound healing processes, and anti-inflammatory effects.

[0130] As used herein, the term "HPR (phthalein oxalate)" is a synthetic retinol derivative that is used in wound repair preparations to promote cell renewal, enhance wound repair ability and improve healing efficiency, while also improving skin aging that may occur during wound healing.

[0131] As used herein, the term "barrier function" refers to a series of physiological functions of the skin that collectively form a defense against external harmful agents. This includes, but is not limited to, preventing pathogens from invading, reducing water evaporation, blocking ultraviolet radiation, and resisting chemical damage. This barrier function is achieved through the coordinated efforts of multiple layers of the skin, including the epidermal stratum corneum, the sebum membrane, and the coordinated balance of the microbiome.

[0132] As used herein, the term "skin barrier formation" refers to the complex biochemical processes by which various cellular and molecular components in the skin, particularly the epidermis, interact to form a protective barrier. This process involves the production, differentiation, and death of keratinocytes, as well as complex interactions with lipids, thereby establishing an effective physical and chemical barrier.

[0133] As used herein, the term "hydration status" refers to the level of water required for the skin to maintain its normal structure and physiological functions. Hydration status affects the skin's appearance, softness, elasticity, and resistance to irritation. The regulation of skin hydration status involves multiple factors, including water exchange within and between stratum corneum cells, the production of natural moisturizing factors, and the absorption and loss of external water.

[0134] As used herein, the term "filagrin (FLG)" refers to a protein of the keratin family found primarily in the stratum corneum of the epidermis. FLG plays a central role in the construction of the skin barrier, responsible for retaining moisture in the stratum corneum and, through its hydrolysis products, maintaining acid-base balance and an antimicrobial environment. FLG deficiency or dysfunction is often associated with various skin diseases, such as eczema and keratosis.

[0135] As used herein, the term "loricrin (LOR)" refers to another structural protein in the stratum corneum of the epidermis. It is a major component of the skin barrier and is responsible for strengthening the skin's structural integrity. LOR provides additional mechanical strength to the skin by cross-linking with other proteins within the stratum corneum and plays a key role in the skin's defense mechanism.

[0136] As used herein, the term "transglutaminase 1 (TGM1)" refers to an enzyme primarily expressed in the stratum granulosum and stratum corneum of the epidermis, which functions to promote the formation of robust cross-links between skin proteins. The enzymatic reaction catalyzed by TGM1 is crucial for the integrity and protective capacity of the skin barrier, and its deficiency has been linked to the development of certain inherited skin diseases.

[0137] As used herein, the term "3D epidermal skin model" refers to a laboratory-created, three-dimensional skin model that mimics the structure and function of human skin. These models typically consist of multiple cell types, including epidermal and dermal cells, and can be used to study skin biology, drug screening, and disease modeling.

[0138] As used herein, the term "immunofluorescence" refers to an experimental technique that uses specific antibodies conjugated to fluorescent markers to localize and quantify target antigens in tissue sections or cell preparations. This technique is extremely useful for visualizing the location and expression of specific proteins within cells, particularly in pathology and cell biology research.

[0139] As used herein, the term "composition" or "preparation" refers to a mixture containing one or more collagens described in the present application and other auxiliary agents, such as physiologically acceptable carriers and excipients, which are intended to promote local application to the wound surface, enhance the bioavailability of the active ingredients, and thus accelerate wound healing and tissue regeneration.

[0140] As used herein, the term "antioxidant" refers to a class of substances that can neutralize free radicals and slow or prevent cell damage, which helps reduce cell damage caused by oxidative stress during wound healing, supports a healthy cellular environment, and promotes rapid wound recovery.

[0141] As used herein, the term "emulsifier" refers to a substance that can help oil and water mix to form a stable emulsion. It is used in wound repair preparations to improve the texture and stability of the product and ensure that the active ingredients are evenly distributed for easy topical application.

[0142] As used herein, the term "humectant" refers to a substance that can attract or lock in moisture and help the skin retain moisture, which is crucial for maintaining a suitable moist environment in the wound treatment area and accelerating cell repair and wound healing.

[0143] As used herein, the term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacture, storage, and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (e.g., arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine, octopine, strombine, and trimethylamine N-oxide (TMAO), human serum albumin (HSA), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play the role of controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salt (such as arginine hydrochloride), glycine, and proline. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, and arginine hydrochloride-sucrose.

[0144] As used herein, the term "type 21 collagen hydrogel dressing" refers to a polymer material formulated based on the patented type 21 collagen and auxiliary ingredients such as hyaluronic acid, which forms a three-dimensional network structure through the use of a chemical crosslinker. This hydrogel dressing plays a key role in wound repair and cell regeneration, enhancing the survival and activity of damaged cells, and exhibiting exceptional cytoprotective effects, particularly under oxidative stress. DETAILED DESCRIPTION

[0145] In a specific embodiment, the materials and methods used are as follows:

[0146] Cell lines: HaCaT (human keratinocytes) and HFF-1 (human fibroblasts) were purchased from Fenghui Biotechnology.

[0147] Reagents: CCK-8 kit (Japan Tongren CK04) was used for cell viability assessment, and AQP3 (Shanghai ELISA) and type I collagen kits (Shanghai ELISA CMM2023H1) were used for related protein expression analysis.

[0148] Example 1: Cell scratch assay

[0149] This example aims to evaluate the role of recombinant humanized collagen type 21 (ColpepA1 21) in promoting wound healing, especially its effect on the migration ability of skin cells. Cell migration is a key process in wound repair and regeneration, and is essential for rapid wound closure and high-quality healing. The experiment used HaCaT keratinocytes and HFF-1 fibroblasts as models to explore how different concentrations of recombinant humanized collagen type 21 promote the migration and healing of these cells, so as to further explore its application potential in the field of wound repair.

[0150] Experimental groups:

[0151] Blank control group (Control): No therapeutic agent was added, and cell migration was directly observed after the scratch test to observe natural cell migration or wound closure speed.

[0152] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added and the effect on cell behavior was evaluated after a scratch test as a known cell migration and healing promoter.

[0153] Collagen control group (Col III): Add 50ppm type III collagen and perform a scratch test as a reference for the effect of the control collagen.

[0154] ColpepA1 21 experimental group: 10ppm and 50ppm of recombinant humanized collagen type 21 (ColpepA1 21) were added, and a scratch test was performed to examine the effects at different concentrations.

[0155] ColpepA1 21 (long chain) experimental group: 50 ppm of type 21 recombinant humanized collagen ColpepA1 21 (long chain) was added to perform a scratch test to examine the difference in effect between it and the patented ColpepA1 21 (short chain).

[0156] Wherein, the COL 21 amino acid sequence is selected from SEQ ID No. 2:

[0157] YVHHHHHHENLYFQGEDGEVRSSCRTAPTDLVFILDGSYSVGPENFEIVKKWLVQITKNFDIGPKFIQ

[0158] VGVVQYSDYPVLEIPLGSYDSGEHLTAAVESILYLGGNTKTGKAIQFALDYLFAKSSRFLTKIAVVLT

[0159] DGKSQDDVKDAAQAARDSKITLFAIGVGSETEDAELRAIANKPSSTYVFYVEDYIAISKIREVMKQK

[0160] LCEESVCPTRIPVAARDERGFDILLGLDVNKKVKKRIQLSPKKIKGYEVTSKVDLSELTSNVFPEGLP

[0161] PSYVFVSTQRFKVKKIWDLWRILTIDGRPQIAVTLNGVDKILLFTTTSVINGSQVVTFANPQVKTLFD

[0162] EGWHQIRLLVTEQDVTLYIDDQQIENKPLHPVLGILINGQTQIGKYSGKEETVQFDVQKLRIYCDPEQ

[0163] NNRETACEIPGFNGECLNGPSDVGSTPAPCICPPGKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQ

[0164] GIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGF

[0165] YGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPG

[0166] NRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG.

[0167] This specific amino acid sequence is derived from patent CN114195884A. This experimental group aims to further validate the effectiveness of ColpepA121 in promoting cell migration. By comparing the effects of known long-chain collagens with the short-chain collagens described in this patent, the authors evaluated the differences in the effects of the short-chain collagens compared to the long-chain collagens on promoting cell migration and wound closure.

[0168] HaCaT keratinocytes were cultured separately and plated for 24 hours. Sample treatments were added when the cells reached 50% to 60% confluency, and wound wounds were performed when the cells reached 100% confluency. The five experimental groups differed in the treatment conditions during the culture process. All groups were scarified and cultured for 36 hours. Photos were taken at 0, 12, 24, and 36 hours to compare the progress of wound healing.

[0169] Data Collection and Analysis:

[0170] 1. Visual Assessment

[0171] like Figure 1 As shown, the blank control group showed very limited cell migration in the scratch area across all time points, indicating a baseline level of cell migration or wound closure rate. The 50 ppm type III collagen group served as a control to understand the effect of standard type III collagen on cell migration. The TGFβ1 group demonstrated enhanced cell migration compared to the blank control, suggesting that TGFβ1 acts as a stimulator of cell migration, consistent with its known biological role in wound healing. The 10 ppm ColpepA121 group showed increased migration compared to the blank control, but this was less than the 50 ppm ColpepA1 21 group, suggesting a dose-dependent response to ColpepA1 21. The 50 ppm ColpepA121 group demonstrated a significant increase in cell migration in the scratch area, with a visually observable decrease in wound width over time. Cell migration is a key step in wound healing, so this finding highlights the potential role of type 21 collagen in accelerating wound closure, which has important implications for the development of wound repair materials.

[0172] 2. Quantitative Analysis

[0173] like Figure 2 The percentage of wound closure at different time points is shown for the blank control group, positive control group, collagen control group, and experimental groups with different concentrations of ColpepA1 21. The bars represent the average percentage of closure, and the error bars indicate the intra-group variation.

[0174] 12 hours: All groups showed some degree of wound closure, with varying degrees of effectiveness. The closure percentage in the 50 ppm ColpepA1 21 group was higher than in the blank control group and the lower concentrations of ColpepA1 21.

[0175] 24 hours: The differences in wound closure rates between the different treatment groups became more apparent, with the 50ppm ColpepA1 21 group showing better healing compared to the blank control and type III collagen groups.

[0176] 36 hours: The 50 ppm ColpepA1 21 group showed the highest wound closure rate, approximately 63.85%, which was significantly higher than the blank control group and the 10 ppm ColpepA1 21 group.

[0177] Quantitative data strongly demonstrated that recombinant humanized collagen type 21 peptide (ColpepA1 21) significantly enhanced the migration and wound closure ability of HaCaT cells, especially at a high concentration of 50ppm. The blank control group served as a baseline for natural wound closure, while the TGFβ1 group confirmed the known positive effect of this growth factor on cell migration. The increased migration in the 50ppm ColpepA1 21 group not only demonstrated the effectiveness of the peptide in promoting wound healing, but also suggested that a dose-dependent mechanism may exist, where higher concentrations lead to greater cell migration.

[0178] Figure 3 The percentage of wound closure after 12 hours is shown for the blank control group, the positive control group, and the experimental groups treated with ColpepA1 21 (50 ppm) and ColpepA1 21 (long chain). At the 12-hour time point, each experimental group demonstrated varying degrees of cell migration and wound closure. The blank control group (Control), serving as a baseline, exhibited the lowest wound closure rate, averaging 3.89%. This result was expected, as the control group was not treated with any collagen or growth factors, reflecting the natural migration ability of the cells. The positive control group (TGFβ1) exhibited an average wound closure rate of 33.93%, a significant improvement consistent with the known role of TGF-β1 in promoting cell migration and healing. The ColpepA1 21 (long chain) experimental group exhibited an average wound closure rate of 28.19%, which, while lower than the TGFβ1-treated positive control group, was significantly higher than the naturally healing blank control group. This suggests that ColpepA1 21 (long chain) can promote cell migration and wound healing, although its effectiveness is slightly lower than that of known growth factors. It is worth noting that the ColpepA121 experimental group of this patent showed an average scratch closure rate of 41.44%, which was significantly higher than that of the blank control group. It was not only higher than the ColpepA1 21 (long chain) experimental group, but also showed a stronger promoting effect than the positive control group.

[0179] The experimental results show that the patented recombinant humanized collagen type 21 (ColpepA1 21) can significantly promote skin cell migration and accelerate wound closure, especially at higher concentrations. Its effect is more obvious, and its effect is better than some known promoting factors. This emphasizes the potential application value of ColpepA1 21 in the development of wound repair materials and provides an important scientific basis for the design and development of new composite materials based on type 21 collagen. In addition, its ability to promote cell migration and accelerate wound healing further confirms the significant advantages of ColpepA1 21 in improving the quality and speed of wound healing, especially in situations where rapid wound healing is required.

[0180] Example 2: Production of Aquaporin 3

[0181] This study aimed to investigate the effect of recombinant humanized collagen type 21 (ColpepA1 21) on the expression of aquaporin 3 (AQP3), a key factor in wound healing. AQP3 is crucial for maintaining skin moisture balance, promoting cell proliferation, and accelerating wound healing. By evaluating the effect of ColpepA1 21 on AQP3 expression, we aimed to reveal its potential for wound repair and skin regeneration.

[0182] Experimental groups:

[0183] Blank control group (Control): No reagent is added and used for comparison of baseline levels.

[0184] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added as a positive control, which is known to promote AQP3 expression.

[0185] Collagen control group (Col III): 50 ppm type III collagen was added to compare the effects of different types of collagen.

[0186] ColpepA1 21 experimental group: 10 ppm of recombinant humanized collagen type 21 was added to evaluate its effect on AQP3 expression.

[0187] All groups cultured HaCaT keratinocytes, plated and cultured for 24 hours. When the cell confluence reached 90%, sample treatment was added and cultured for 24 hours. The cells were then harvested and lysed on ice. After high-speed centrifugation (10,000 rpm), the supernatant was collected and used to detect the AQP3 expression level using an ELISA kit.

[0188] Data Collection and Analysis:

[0189] The sample is added to a microplate pre-coated with a specific antibody, followed by the addition of a secondary antibody that binds to the target protein. Finally, the addition of a substrate produces a color reaction whose intensity is proportional to the protein content in the sample. The intensity of the color reaction in each well is read using a microplate reader, and the specific AQP3 content is calculated using a standard curve.

[0190] Figure 4 The results show the aquaporin 3 (AQP3) levels in each experimental group. The blank control group (Control) exhibited the lowest AQP3 expression level, providing a baseline for AQP3 levels in untreated cells. The TGFβ1 group significantly increased AQP3 levels, consistent with TGFβ1's role in promoting cell proliferation and migration in cell biology, suggesting that TGFβ1 can effectively increase AQP3 expression. The 50 ppm type III collagen group (Col III) also increased AQP3 levels, but the effect appeared to be less pronounced than that of the TGFβ1 group. The 10 ppm recombinant humanized collagen type 21 group (ColpepA1 21) increased AQP3 levels by 16.93% compared to the blank control group, demonstrating that this low concentration of ColpepA1 21 can promote AQP3 expression.

[0191] In the field of wound repair, maintaining appropriate wound moistness is considered to be one of the key factors in promoting healing. Since AQP3 plays a core role in regulating skin water transfer and maintaining cellular water balance, its increased expression is extremely important for promoting rapid wound healing and maintaining the hydration state of damaged skin. This is of great significance for maintaining the appropriate moisture state of the wound surface and accelerating cell migration and proliferation during the wound healing process. The results of this example show that ColpepA1 21 can significantly promote the expression of AQP3 even at low concentrations, proving that it has a highly effective promoting effect. This is particularly important for the development of wound repair materials with high dosage efficiency and good cost-effectiveness. This example demonstrates the potential value of ColpepA1 21 in promoting wound healing and enhancing skin barrier function.

[0192] Therefore, this example not only demonstrates the application value of recombinant humanized collagen type 21 in wound repair and skin regeneration, but also provides a solid scientific basis for further research on the potential mechanisms of ColpepA1 21 in wound repair. This discovery is expected to promote the development of new composite materials based on type 21 collagen, providing more effective treatment options for wound repair.

[0193] Example 3: 3D epidermal skin model test

[0194] This example aims to evaluate the effect of type 21 collagen (ColpepA1 21) on wound repair in a 3D epidermal skin model by measuring changes in the levels of filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1). These three proteins play a key role in the formation and maintenance of the skin barrier, and changes in their levels can directly reflect improvements in skin repair and barrier function.

[0195] The experimental groups are as follows:

[0196] Blank control group (BC): No treatment or medication was performed, used to provide baseline data for the experiment.

[0197] Negative control group (NC): received 600mJ / cm 2 UVB irradiation without any drug treatment is used to demonstrate the damaging effect of UVB irradiation.

[0198] Positive control group (PC): received 600mJ / cm 2 UVB irradiation and WY14643 (50uM) treatment were performed. WY14643 is a known repair promoter to demonstrate the effectiveness of the experimental system response.

[0199] Sample group (Collagen type 21 group, ColpepA1 21): received 600mJ / cm 2 The cells were irradiated with UVB and treated with 0.0125% (v / v) type 21 collagen to evaluate its repair effect on UVB-induced damage.

[0200] Test system: 3D epidermal skin model The batch number is ES231107, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0201] Main reagents: EpiGrowth culture medium (Guangdong Boxi Biological), PBS (Solaibao), WY14643 (Sigma), FLG antibody (Abeam), LOR antibody (Abeam), TGM1 antibody (Abeam), paraformaldehyde (Biosharp).

[0202] Main equipment: CO2 incubator (Thermo, 1501), clean bench (Suzhou Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500).

[0203] Experimental process:

[0204] 1. Model preparation: Transfer the 3D epidermal skin model to a 6-well plate, add 0.9 mL of EpiGrowth culture medium to each well, and mark the test group number on the 6-well plate.

[0205] 2. Sample treatment: According to the grouping, evenly apply the sample solution on the model surface. For all groups that need UVB irradiation, 600mJ / cm 2 The model was then placed in a CO2 incubator and incubated at 37°C and 5% CO2 for 24 hours.

[0206] 3. Model cleaning: After incubation, use sterile PBS to clean the surface of the model to remove residual test substance, and then use a sterile cotton swab to gently wipe away the residual liquid inside and outside the model.

[0207] 4. Immunofluorescence Testing: After model processing and incubation, the model to be tested was circumcised and fixed with 4% paraformaldehyde for 24 hours. After fixation, immunofluorescence detection of FLG, LOR, and TGM1 was performed. Images were taken using a fluorescence microscope, and relevant image data was collected for analysis.

[0208] 5. Calculation of the improvement rate: Based on the data obtained from immunofluorescence detection, the improvement rate of each experimental group was calculated using the following formula to evaluate the repair effect of ColpepA1 21:

[0209] Improvement rate (%) = (experimental group - blank control group) / blank control group × 100%

[0210] Figure 5 The relative integrated optical density (IOD) values ​​of FLG are shown, reflecting the expression levels of FLG under different experimental conditions. T-test was used to test the significant differences between the corresponding experimental groups and the negative control group, where significance is indicated by *, * indicates p < 0.05, and ** indicates p < 0.01 ( Figure 6 Figure 7 Same). FLG is an indispensable component of the stratum corneum structure, which can promote skin hydration and enhance barrier function. The hydrolyzate of FLG is a natural moisturizing factor that can effectively retain skin moisture, maintain skin barrier function and the overall health of the skin. The basal control group (BC) had the highest FLG expression, indicating the state of normal skin barrier function. In contrast, the IOD value of FLG in the negative control group (NC) decreased significantly after UVB irradiation, revealing the effect of UVB irradiation on keratinocytes and the damage to barrier function induced by it. In the positive control group (PC), after treatment with WY14643, its FLG expression increased significantly, thus confirming the repair effect of the positive drug on stratum corneum damage. At the same time, the application of type 21 collagen (ColpepA1 21 0.0125%) in the sample group significantly increased FLG expression, suggesting its potential role in promoting stratum corneum recovery and its barrier function.

[0211] Figure 6 The relative integrated optical density (IOD) values ​​of loricrin (LOR) are shown. LOR is one of the main proteins in epidermal keratinocytes and constitutes the barrier of the outer layer of the skin. It strengthens the structural strength and barrier function of the skin by cross-linking with other proteins, and produces the ability to resist external physical, chemical and biological stimuli. Under the influence of UVB irradiation, the negative control group (NC) showed a significant decrease in LOR expression, indicating that external stimuli have damaged the structural strength and barrier function of the skin. After the positive control group (PC) was treated with WY14643, the LOR expression was significantly increased, indicating the role of positive drugs in the restoration of the barrier function of the stratum corneum. The LOR level of the sample group type 21 collagen (ColpepA1 21 0.0125%) was significantly higher than that of the NC group, which further emphasized the importance of type 21 collagen in improving the skin barrier and defense capabilities, which is especially critical for wound healing.

[0212] Figure 7 The relative integrated optical density (IOD) values ​​of transglutaminase 1 (TGM1) are shown. TGM1 is a key enzyme that participates in the cross-linking process of skin stratum corneum proteins, promotes the terminal differentiation of keratinocytes, and forms a strong skin barrier. Its activity is essential for maintaining the integrity and function of the skin barrier. As shown in the figure, the TGM1 level of the negative control group (NC) was significantly reduced by UVB irradiation, highlighting the negative effect of irradiation on the cross-linking process of stratum corneum proteins. The positive control group (PC) showed a significantly increased TGM1 level compared with the negative control group, indicating that WY14643 may promote the expression of TGM1. The significant promoting effect of type 21 collagen (ColpepA1 21 0.0125%) in the sample group on TGM1 (the increase rate was 315.63%) revealed its positive effect on accelerating skin barrier formation and terminal differentiation of keratinocytes, which has obvious benefits for rapid wound recovery and reducing the risk of infection.

[0213] In summary, the results of this study clearly indicate that type 21 collagen (ColpepA1 21) has a significant positive regulatory effect on the expression of skin barrier proteins FLG, LOR, and TGM1. These proteins are key components of skin moisturizing, structural integrity, and barrier function, and their increased expression is crucial for skin repair and wound healing after UVB irradiation. The application of type 21 collagen can promote the restoration of these key factors, provide necessary barrier protection for wounds, and may accelerate the healing process. Therefore, type 21 collagen not only demonstrates its potential clinical application prospects in the fields of skin biology and wound treatment, but also provides a valuable ingredient basis for the development of skin care products. These findings support further clinical research and product innovation to translate these benefits of type 21 collagen into effective strategies for wound treatment.

[0214] Based on the above experimental results, recombinant humanized collagen type 21 (ColpepA1 21) showed significant promoting effects in key biological links of the wound repair process, providing comprehensive support for wound healing and skin regeneration:

[0215] Promoting Cell Migration: ColpepA1 21, particularly at a higher concentration (50 ppm), demonstrated significant wound healing acceleration in the HaCaT cell scratch assay, which is crucial for rapid wound closure and shortened healing cycles. At the dermal callus level, ColpepA1 21 effectively promoted cell migration, a key step in the wound repair process.

[0216] Enhanced Hydration: ColpepA1 21 significantly increases AQP3 expression, highlighting its importance in maintaining a hydrated environment within the wound and surrounding tissues, and in promoting normal cellular function during wound healing. This enhances the hydration capacity of skin cells. During wound repair, increased AQP3 expression helps maintain optimal hydration within the wounded area, thereby promoting normal cellular function and effective wound healing.

[0217] Strengthening the rapid formation of the epidermal barrier: ColpepA121 revealed its potential role in promoting keratinocyte migration and shortening the healing cycle by significantly increasing the expression of the key protein FLG. In addition, the enhanced expression of LOR demonstrated the effectiveness of ColpepA121 in strengthening the epidermal barrier structure and improving the skin's defense against external stimuli. Furthermore, the increase in TGM1 activity emphasizes the key role of ColpepA121 in the terminal differentiation process of keratinocytes and the formation of the epidermal barrier.

[0218] Together, these experimental results highlight the potential of ColpepA1 21 in wound repair, particularly as a key active ingredient in novel collagen composite materials. ColpepA1 21 not only promotes rapid skin repair and restoration of structural integrity, but also enhances the skin's natural barrier function and hydration status. These properties make it an ideal candidate for the development of effective wound repair strategies and skin regeneration products. Therefore, ColpepA1 21 is expected to become a key factor in advancing skin health and repair science in the future development of wound repair materials and skin care products.

[0219] Example 4: Preparation of Type 21 Collagen ColpepA1 21 Hydrogel Dressing

[0220] Material:

[0221] Type 21 collagen ColpepA1 21 solution: 3% (w / v)

[0222] Hyaluronic acid: 1% (w / v)

[0223] Cross-linking agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC): 0.1% (w / v)

[0224] Mixing steps:

[0225] Dissolve collagen and hyaluronic acid separately in sodium acetate buffer (pH 5.5) at room temperature. Slowly add the hyaluronic acid to the collagen solution, stirring continuously to ensure uniform mixing. Slowly add EDC as a crosslinker to initiate the crosslinking reaction. Continue stirring at room temperature for 1 hour to promote sufficient crosslinking and form a preliminary crosslinked network.

[0226] Gel formation:

[0227] The mixture was transferred to a mold and allowed to stand at room temperature for 1 hour. The mold was then refrigerated at 4°C for 24 hours to promote complete gelation. The gel was removed from the mold and soaked in sterile cold saline for 12 hours to remove unreacted crosslinker and byproducts.

[0228] Sterilization and packaging:

[0229] Use low-temperature sterilization methods such as plasma sterilization technology to reduce potential thermal damage. Under aseptic operating conditions, cut the hydrogel into the required size, seal it in sterile packaging materials, and store it at 4°C, away from light and high temperature to maintain the stability and biological activity of the product and prevent microbial contamination and drying.

[0230] Comparative Example 1: Preparation of Type III Collagen Hydrogel Dressing

[0231] Material:

[0232] Type III collagen solution: 3% (w / v)

[0233] Hyaluronic acid: 1% (w / v)

[0234] Cross-linking agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC): 0.1% (w / v)

[0235] The preparation method is the same as Example 4.

[0236] Example 5: Experiment on promoting wound healing and cell regeneration with type 21 collagen hydrogel dressing

[0237] This study aimed to evaluate the effects of type 21 collagen hydrogel dressings on barrier keratinocyte activity under cell culture conditions, specifically their protective effects against oxidative stress. By comparing this with type III collagen hydrogel dressings, the potential of type 21 collagen in promoting wound healing and cell regeneration was further explored.

[0238] Experimental methods:

[0239] Cell seeding: Culture keratinocytes HaCaT and dilute the cells to 3×10 5 The cell dilution was inoculated into a 96-well plate at a seeding density of 100 μl / well and incubated in a cell culture incubator (37° C., 5% CO 2 , 95% RH) for 24±2 h.

[0240] Experimental groups:

[0241] Normal group (Control) and zero-adjustment group: containing only culture medium;

[0242] H2O2 modeling group (NC): cells were treated with H2O2 to simulate oxidative stress;

[0243] The sample groups are based on the H2O2 modeling group, and the conditions are as follows:

[0244] Sample group A: added 1000ppm ColpepA1 21;

[0245] Sample Group B: Type III collagen hydrogel prepared using the method of Comparative Example 1 with the addition of an equal amount of 1000 ppm type III collagen;

[0246] Sample group C: type 21 collagen mixed hydrogel prepared using the method of Example 4, to which an equal amount of 1000 ppm of ColpepA1 21 was added;

[0247] The activity test experiment was set up with zero adjustment group, normal group, H2O2 model group and sample group. Each group was set up with three replicate wells.

[0248] Liquid preparation:

[0249] Sample: Dissolve the sample with cell culture medium (add DMSO to dissolve the sample if it cannot be dissolved, but the final DMSO concentration should not exceed 0.5%), and then dilute it with cell culture medium; H2O2 working solution: Prepare H2O2 working solution from H2O2 stock solution using serum-free culture medium.

[0250] CCK-8 working solution: Add 1 ml of CCK-8 stock solution to 9 ml of serum-free culture medium and mix well to prepare 10 ml of CCK-8 working solution.

[0251] H2O2 induction: When the cell confluence in the 96-well plate reached 50%-70%, the cell culture medium in each group was aspirated, the cells were washed once with PBS, discarded, and H2O2 working solution was added.

[0252] Sample addition: After induction, the plates were washed twice with PBS. 100 μl of culture medium was added to each well of the normal group; 100 μl of culture medium containing samples of corresponding concentrations was added to each well of the sample group; the zero-adjustment group had no cell inoculation and only 100 μl of cell culture medium was added. After sample addition, the 96-well plate was placed in an incubator (37°C, 5% CO2, 95% RH) and cultured for 24 ± 2 h.

[0253] Cell viability assay: After culturing cells for 24 ± 2 h, observe the cell status under a microscope, discard the supernatant, add 100 μl of CCK-8 working solution to each well of each group, and incubate at 37°C in the dark for 2 ± 0.5 h. After incubation, read the OD value at 450 nm and calculate the percentage of cell viability according to the following formula.

[0254] Cell activity (%) = (OD of sample group - OD of zero-adjusted group) / (OD of blank group - OD of zero-adjusted group) × 100%.

[0255] Figure 8 The cell activity data of the normal group (Control), H2O2 modeling group and three sample groups are shown. The results show that the normal group serves as the baseline, indicating the cell survival state in a natural state without any external intervention. The cell activity of the H2O2 modeling group dropped significantly to 71.16%, indicating that significant oxidative damage occurred under the oxidative stress environment, reducing the activity of the cells. The cell activity of sample group A with the addition of 1000ppm ColpepA121 rose to 119.26%, indicating that type 21 collagen can significantly increase the activity of damaged cells, exceeding the level under normal physiological conditions. The cell activity of sample group B with the addition of 1000ppm type III collagen mixed hydrogel was 111.22%, which also showed a certain alleviating effect on oxidative damage, but was slightly inferior to pure type 21 collagen. The cell activity of sample group C with the addition of 1000ppm type 21 collagen mixed hydrogel was the highest, reaching 145.89%, which indicates that the composite material of type 21 collagen and other components has a synergistic effect, further improving the recovery and survival ability of cells.

[0256] The high cell activity of type 21 collagen hydrogel indicates that it can not only protect cells from oxidative stress damage caused by H2O2, but also promote the recovery of damaged cells. In particular, the highest cell activity shown in sample group C suggests that ColpepA1 21 may trigger cell signaling pathways in specific formulations, thereby enhancing the cells' antioxidant and repair capabilities. These properties of type 21 collagen hydrogel dressings, especially its excellent effects shown at higher concentrations and in composite materials, provide strong scientific support for its clinical application in treating inflammation and promoting wound healing. Due to its excellent cell activity-promoting effect, type 21 collagen hydrogel can be used as a potential therapeutic material to promote wound healing, reduce inflammatory responses, and accelerate skin regeneration.

[0257] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. It will be apparent to those skilled in the art that any equivalent modifications and substitutions to the present invention fall within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are encompassed within the scope of the present invention.

Claims

1. A method for preparing a collagen material for wound repair, wherein: The method steps are selected from any one of the following methods (1) to (4): Method (1): A mixed material prepared by mixing collagen and auxiliary ingredients in a sodium acetate buffer solution is subjected to cross-linking reaction at room temperature; the mixed solution is transferred to a mold, allowed to stand, and refrigerated for 12-24 hours to form the mold; Low temperature curing, plasma sterilization; or Method (2): Pre-soak collagen and auxiliary ingredients in water and heat to dissolve; add hyaluronic acid solution, stir and solidify at room temperature; solidify at low temperature and sterilize with plasma; or Method (3): Mix collagen and auxiliary components, spin-coat on substrate; dry and peel, and sterilize by gamma irradiation; or Method (4): Mix collagen and auxiliary ingredients, pour into a mold, freeze and then freeze-dry; Gas cross-linking, gamma ray sterilization; The collagen comprises type 21 recombinant humanized collagen, and the amino acid sequence of the collagen comprises the amino acid sequence shown in SEQ ID No.

1.

2. The method according to claim 1, wherein The auxiliary ingredients are selected from one or more of the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, and thickeners.

3. The method according to claim 1 or 2, wherein The weight ratio of the collagen and auxiliary ingredients is selected from: Collagen and antimicrobial agent: 5:1~20:1; or Collagen and anti-inflammatory agent: 20:1~100:1; or Collagen and growth factor: 50:1~200:1; or Collagen and moisturizer: 1:1~10:1; or Collagen and antioxidants: 10:1~50:1; or Collagen and emulsifier or thickener: one or more combinations in a ratio of 5:1 to 20:

1.

4. The method according to claim 1 or 2, wherein: The method steps are selected from any one of the following methods (1) to (4): Method (1): a mixture of type 21 collagen, hyaluronic acid, and a cross-linking agent is subjected to cross-linking reaction in sodium acetate buffer at room temperature; the mixture is transferred to a mold, allowed to stand, and refrigerated for 12-24 hours to form; low-temperature curing and plasma sterilization; or Method (2): pre-soak the mixture of gelatin and collagen in water and heat to dissolve; add hyaluronic acid solution, stir and solidify at room temperature; solidify at low temperature and sterilize by plasma; or Method (3): Mix type 21 collagen and PLGA in a certain proportion, spin-coat on the substrate, dry and peel, and sterilize by gamma ray; or Method (4): Mix type 21 collagen and chitosan in a certain proportion, pour into a mold, freeze, and then freeze-dry; Gas cross-linking and gamma ray sterilization.

5. The method according to claim 4, wherein The ratio in the method (1) is: 2-3% (w / v) type 21 collagen, 1% (w / v) hyaluronic acid, and 0.1% (w / v) cross-linking agent.

6. The method according to claim 5, wherein: The ratio of type 21 collagen is 3% (w / v).

7. The method according to claim 4, wherein: In the method (1), the cross-linking agent is EDC.

8. The method according to claim 4, wherein: The ratio in the method (2) is: 2% (w / v) type 21 collagen solution, 1% (w / v) hyaluronic acid, and 3% (w / v) gelatin.

9. The method according to claim 4, wherein: The ratio in the method (3) is: 1.5% (w / v) type 21 collagen solution, 2% (w / v) PLGA.

10. The method according to claim 4, wherein: The ratio in the method (4) is: 2.5% (w / v) type 21 collagen solution and 1.5% (w / v) chitosan.

11. A collagen material prepared according to the method according to any one of claims 1 to 10, wherein The collagen material is used for repairing wounds after skin damage.

12. A medical composition, wherein The composition comprises the collagen material prepared by the method according to any one of claims 1 to 10 or according to claim 11.

13. The medical composition according to claim 12, wherein The composition is a solid, liquid, semi-solid or gel preparation.

14. The medical composition according to claim 12, wherein The composition is in the form of a preparation selected from one or more of a smearable gel, a cream, a dressing, a spray, an oral medication or an injection.

15. The medical composition according to claim 14, wherein The composition is in the form of a dressing, including but not limited to a hydrogel dressing, a film dressing, a sponge or a fiber dressing.

16. The medical composition according to claim 15, wherein The composition is in the form of a hydrogel dressing.

17. The medical composition according to claim 16, wherein The composition is in the form of a collagen-hyaluronic acid hydrogel dressing.

18. Use of type 21 recombinant humanized collagen in the preparation of a medical composition for wound repair, wherein: The amino acid sequence of the collagen comprises the amino acid sequence shown in SEQ ID No. 1, and the medical composition comprises the collagen material prepared by the method according to any one of claims 1 to 10 or according to claim 11.

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