Novel composite collagen material for wound repair

Abstract

The invention relates to the field of biomedical materials, in particular to a novel composite collagen material for wound repair, and particularly relates to a preparation method of the novel composite collagen material containing 21-type recombinant humanized collagen (ColpepA1 21) and application of the novel composite collagen material in wound repair. The ColpepA1 21 is used for remarkably promoting key biological reactions in the wound healing process, including accelerating cell migration, enhancing the skin hydration state and barrier function and improving the stability and elasticity of the skin structure, a novel composite collagen material is developed, wound healing and cell regeneration can be effectively promoted, and oxidative damage can be relieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a method for preparing a novel composite collagen material for wound repair and its application in wound repair, including a method for preparing 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 more than 30% of the total protein in the human body, but also the cornerstone for maintaining the structure and function of various tissues in the human body. It exists in the skin, bones, tendons, blood vessels and other connective tissues of the human body, forming a three-dimensional network structure that provides mechanical strength and stability of the tissue. In the skin, collagen is responsible for maintaining the firmness and elasticity of the skin and keeping the skin surface smooth and shiny. With age, the natural production rate of collagen in the human body begins to slow down, and the existing collagen will gradually degrade and be damaged due to factors such as ultraviolet radiation, environmental pollution, and bad living habits. This causes the skin to gradually lose its original elasticity and firmness, and signs of aging such as sagging, fine lines and wrinkles appear, as well as a decrease in wound healing ability. In addition, the reduction of collagen in other tissues will also affect the density of bones, the elasticity of tendons, and the stability of blood vessels, thereby affecting overall health. In the field of wound repair, traditional treatment methods face challenges such as 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 outcomes and alleviate patient pain remains an important research direction.

[0003] In this context, it is particularly important to conduct in-depth research on specific types of collagen, such as type 21 collagen (COL21A1). 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. Compared with other more well-known types of collagen, type 21 collagen has been relatively less studied. Type 21 collagen 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 extracellular matrix components. According to the research of Chou MY and Li HC [Chou MY, Li HC (March 2002)], type 21 collagen is mainly expressed in vascular smooth muscle cells and may play a role in the assembly of the extracellular matrix during angiogenesis. In addition, the gene expression of type 21 collagen is regulated by the developmental stage, and the expression level is higher in the fetal stage, 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 function in vascular health and blood pressure regulation. Surendran et al. (2016) found through a large-scale genotype survey that the type 21 collagen gene is involved in the formation of collagen in various tissues such as the heart and aorta, and is associated with vascular remodeling and hypertension. This provides a new perspective for the study of COL21A1 in promoting skin health and anti-aging. These characteristics give type 21 collagen unique application potential in the fields of skin health, anti-aging and vascular health.

[0004] Although research on type 21 collagen in vascular health and blood pressure regulation has been involved, 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 provide a more effective, safe and biocompatible treatment plan for wound repair by in-depth research on the biological characteristics 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 and 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 the collagen material into a shape; 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, 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 described in 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: subjecting a mixed material prepared by proportioning collagen and auxiliary ingredients to a cross-linking reaction in a sodium acetate buffer at room temperature; transferring the mixed solution to a mold, allowing the mixture to stand, and refrigerating the mold for 12-24 hours to form the mixture; 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 gamma ray sterilization.

[0018] In one embodiment, the method of the present invention comprises the following steps: mixing collagen and auxiliary components, 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, etc.

[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: 20:1~100:1; or

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

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

[0027] Collagen and 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: subjecting a mixed material composed of type 21 collagen, hyaluronic acid and a cross-linking agent to a cross-linking reaction in a sodium acetate buffer at room temperature; transferring the mixed solution to 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, and 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; and low-temperature solidification 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 into a mold, freezing and then freeze-drying; 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 injury.

[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 described in 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, maintain 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, and promote 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 also 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 can 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 wound repair 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 antibacterial agent is selected from 5:1 to 20:1; an appropriate concentration of antibacterial 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 at small doses, while excessive doses 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 added 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 an appropriate amount 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, surgical incisions, burns, abrasions, or chronic wounds.

[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, and the hydrogel dressing 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, aseptic 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 strip, 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, 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 effects 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, and the like.

[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 one of the aforementioned collagen materials in the preparation of a medical composition for wound repair.

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

[0102] 1. Rarity and special effects: Type 21 collagen is relatively rare in the adult human body and has been less studied. Moreover, according to research, its potential applications in the fields of skin health and vascular health are unique. The use of this rare type of collagen to develop wound repair materials demonstrates the innovation of exploring new biomaterials.

[0103] 2. Promote natural skin healing and HaCaT cell migration: Type 21 collagen accelerates the natural healing process of the skin 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 point of 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 the expression of AQP3, which not only helps to maintain moisture in the wound, but also may promote more effective cell migration and wound healing.

[0105] Advantages of the 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 the quality of repair: Promote the production of type I and type III collagen, help build new skin tissue with stable structure and normal function, and improve the quality of repair.

[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 ability to retain moisture 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 the scratch test, including the closure of the scratch at different time points.

[0111] Figure 2 and Figure 3 A comparison of the healing rates of keratinocyte scratches is shown, demonstrating the quantitative analysis results of the healing progress of different experimental tissues.

[0112] Figure 4 The content of aquaporin 3 in cells after different treatments is shown, and the effect of ColpepA1 21 on AQP3 expression is demonstrated 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 difference between the corresponding experimental group 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 The effect of ColpepA1 21 hydrogel dressing on cell activity under oxidative stress environment is shown.

[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, which is widely present in various tissues and organs, such as skin, bones, tendons, ligaments and cornea. The main function of these proteins is to provide strength, stability and elasticity to body tissues, ensuring the normal functioning of various physiological functions. There are at least 28 known types of collagen, each of which plays a unique role and function in the body, forming a complex and finely regulated biological system.

[0121] As used herein, the term "collagen type 21 (COL21A1)" refers to a type of collagen present in the human body, which belongs to the family of small molecule collagens. As part of the FACIT (fibril-associated collagen with interrupted triple helix) family, collagen type 21 plays a vital role in connecting extracellular matrix components. This collagen, through its unique molecular structure, promotes interactions and links 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 collagen types I and III, it plays a role in specific tissues and physiological processes, involving the composition of the extracellular matrix and the mechanism of skin repair. Although collagen type 21 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 the structure of the extracellular matrix 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 obtained by genetic recombination technology that simulates the type 21 collagen naturally produced by the human body. The protein has a specific amino acid sequence encoding, which is intended to promote wound healing, support the formation of new tissue and improve 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, etc., and is considered a valuable research tool because of its ability to 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 a human fetus, which are an important component of the skin structure, responsible for the synthesis of 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 mainly expressed in skin cells and is responsible for regulating water transport in cells, 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 one of the main structural proteins of the skin, which are responsible for maintaining the strength, elasticity and firmness of the skin, respectively. Type I collagen is the most abundant type of collagen and is mainly found in the skin, bones and connective tissues; type III collagen is mainly found in the intercellular matrix of the skin and plays an important role in skin elasticity and repair processes.

[0129] As used herein, the term "TGFβ1" (transforming growth factor β1) refers to a multifunctional cytokine that is able to regulate 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 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, which together form a line of defense to protect the body from external harmful substances. This includes but is not limited to preventing pathogen invasion, reducing water evaporation, blocking ultraviolet radiation, and resisting damage from chemicals. The barrier function is achieved by the coordinated action of multiple layers of the skin, including the stratum corneum of the epidermis, the sebum membrane, and the coordinated balance of the microbial community.

[0132] As used herein, the term "skin barrier formation" refers to the process by which various cellular and molecular components in the skin, especially the epidermis, interact to form a protective barrier through complex biochemical processes. This process includes 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 state" refers to the level of water required for the skin to maintain its normal structure and physiological function. The hydration state affects the appearance, softness, elasticity, and resistance to irritation of the skin. The regulation of the hydration state of the skin involves multiple factors, including the exchange of water 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 "filament aggregate protein (FLG)" refers to a protein of the keratin family, which is mainly found in the stratum corneum of the epidermis. FLG plays a central role in the construction of the skin barrier, is responsible for retaining the moisture of the stratum corneum, and maintains the acid-base balance and antimicrobial environment through its hydrolysis products. The absence or dysfunction of FLG is often associated with a variety of 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, which is a major component of the skin barrier and is responsible for reinforcing the structural integrity of the skin. 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 that is primarily expressed in the stratum granulosum and stratum corneum of the epidermis and functions to promote the formation of a stable cross-linked structure between skin proteins. The enzyme reaction catalyzed by TGM1 is essential for the integrity and protective ability of the skin barrier, and its deficiency is associated with the occurrence of certain hereditary skin diseases.

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

[0138] As used herein, the term "immunofluorescence detection" refers to an experimental technique that uses specific antibodies combined with fluorescent markers to locate and quantify target antigens in tissue sections or cell preparations. This technique is extremely useful in visualizing the location and expression of specific proteins within cells, especially 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 adjuvants, 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 down 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 essential 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 (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-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 RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in 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 by using a chemical cross-linking agent. This hydrogel dressing plays a key role in wound repair and cell regeneration, can improve the survival and activity of damaged cells, and especially shows excellent cell protection effects 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 activity assessment, AQP3 (Shanghai ELISA) and type I collagen kit (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 closure and high-quality healing of wounds. 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 the cell migration was directly observed after the scratch test to observe the 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 the scratch assay as a known cell migration and healing promoter.

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

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

[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 amino acid sequence of COL 21 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 verify the effectiveness of ColpepA121 in promoting cell migration. By comparing the effects of known long-chain collagen and the short-chain collagen of this patent, the difference in the effect of short-chain collagen on promoting cell migration and wound closure rate compared with long-chain collagen is evaluated.

[0168] Keratinocytes HaCaT were cultured separately, plated and cultured for 24 hours, and sample treatment was added when the cell confluence was 50% to 60%, and scratching was performed when the cell confluence was 100%. The difference between the five groups of experiments was that there were differences in the conditional treatment during the culture process. All groups were scratched and cultured for 36 hours, and photos were taken at 0 hours, 12 hours, 24 hours and 36 hours to compare the progress of scratch 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 throughout the time points, indicating a baseline level of cell migration or wound closure speed. The 50ppm type III collagen group was used as a control to understand the effect of standard type III collagen on cell migration. The TGFβ1 group showed enhanced cell migration compared to the blank control group, suggesting that TGFβ1 acts as a stimulator of cell migration, which is consistent with its known biological role in wound healing. The 10ppm ColpepA121 group showed an increase in migration compared to the blank control group, but less than the 50ppm ColpepA1 21 group, suggesting a dose-dependent response to ColpepA1 21. The 50ppm ColpepA121 group showed a significant increase in cell migration in the scratch area, with a visually observable decrease in the width of the scratch over time. Cell migration is one of the key steps in wound healing, so this finding emphasizes the potential role of type 21 collagen in accelerating the wound closure process, which is of great significance for the development of wound repair materials.

[0172] 2. Quantitative Analysis

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

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

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

[0176] 36 hours: The 50ppm ColpepA1 21 group showed the highest wound closure rate, about 63.85%, which was significantly higher than the blank control group and the 10ppm 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 the 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 there may be a dose-dependent mechanism, where higher concentrations lead to greater cell migration.

[0178] Figure 3 The percentage of scratch closure after 12 hours for the blank control group, the positive control group, and the ColpepA1 21 experimental group and the ColpepA1 21 (long chain) experimental group at a concentration of 50 ppm is shown. At the 12-hour time point, each experimental group showed different degrees of cell migration and wound closure effects. The blank control group (Control) was used as a baseline and showed the lowest scratch closure rate, averaging 3.89%. This result was in line with expectations because the control group was not treated with any collagen or growth factors, reflecting only the natural migration ability of the cells. The positive control group (TGFβ1) showed an average scratch closure rate of 33.93%, a significant improvement consistent with the known promotion of TGF-β1 in cell migration and healing. The average scratch closure rate of the ColpepA1 21 (long chain) experimental group was 28.19%, which was lower than the positive control group treated with TGFβ1, but significantly higher than the blank control group of natural healing. This shows that ColpepA1 21 (long chain) can promote cell migration and wound healing, although its effect 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 results of this experiment 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, 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 when it is necessary to quickly promote wound healing.

[0180] Example 2: Generation of Aquaporin 3

[0181] This example aims to explore the effect of recombinant humanized collagen type 21 (ColpepA1 21) on the expression of aquaporin 3 (AQP3), a key factor in promoting wound healing. AQP3 is essential for maintaining skin moisture balance, promoting cell proliferation, and rapid wound healing. By evaluating the promoting effect of ColpepA1 21 on AQP3 expression, it aims to reveal its application potential in wound repair and promoting 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 known to promote AQP3 expression.

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

[0186] ColpepA1 21 experimental group: 10 ppm of type 21 recombinant humanized collagen 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 taken and lysed on ice. After high-speed centrifugation (10,000 rpm), the supernatant was taken and used for the detection of AQP3 expression level by ELISA kit.

[0188] Data Collection and Analysis:

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

[0190] Figure 4 The content of aquaporin 3 (AQP3) in each experimental group is shown. The results show that the blank control group (Control) showed the lowest AQP3 expression level, providing the baseline content of AQP3 in untreated cells. The TGFβ1 group significantly increased the content of AQP3, which is consistent with the role of TGFβ1 in promoting cell proliferation and migration in cell biology, indicating that TGFβ1 can effectively increase AQP3 expression. The 50ppm type III collagen group (Col III) also increased the AQP3 content, but its effect seemed to be lower than that of the TGFβ1 group. Compared with the blank control group, the 10ppm type 21 recombinant humanized collagen group (ColpepA1 21) increased the AQP3 content by 16.93%, indicating that this low concentration of ColpepA1 21 can promote the expression of AQP3.

[0191] In the field of wound repair, maintaining appropriate wound moisture 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 wound healing. The results of this example show that ColpepA1 21 can significantly promote the expression of AQP3 even at low concentrations, proving that it has an efficient 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 studying the potential mechanism of ColpepA1 21 in the field of wound repair. This discovery is expected to promote the development of new composite materials based on type 21 collagen and provide 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, specifically by measuring the changes in the content 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 content can directly reflect the improvement of 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 given. 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 mice 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 (Solebo), WY14643 (Sigma), FLG antibody (Abeam), LOR antibody (Abeam), TGM1 antibody (Abeam), and 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 in advance, and mark the test group number on the 6-well plate.

[0205] 2. Sample treatment: According to the grouping, apply the sample solution evenly 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 the residual test substance, and then use a sterile cotton swab to gently wipe off the residual liquid inside and outside the model.

[0207] 4. Immunofluorescence test: After model processing and incubation, the model for detection was cut into rings and fixed with 4% paraformaldehyde for 24 hours. After fixation, immunofluorescence detection of FLG, LOR, and TGM1 was performed, and photos were taken and observed using a fluorescence microscope, and relevant image data were collected for analysis.

[0208] 5. Calculation of the improvement rate: The data obtained by immunofluorescence detection were used to calculate the improvement rate of each experimental group 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 maintain 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 barrier function damage 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 enhances 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 the damage of external stimuli to the structural strength and barrier function of the skin. After the positive control group (PC) was treated with WY14643, the expression of LOR was significantly increased, indicating the role of positive drugs in the restoration of stratum corneum barrier function. The LOR level of the sample group type 21 collagen (ColpepA1 21 0.0125%) group was significantly higher than that of the NC group, which further emphasized the importance of type 21 collagen in improving 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 involved in the cross-linking process of skin stratum corneum proteins, promoting the terminal differentiation of keratinocytes and forming 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 promotion 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 the formation of the skin barrier and the terminal differentiation of keratinocytes, which has obvious benefits for the rapid recovery of the wound surface 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 essential for skin repair and wound healing after UVB irradiation. The application of type 21 collagen can promote the recovery of these key factors, provide the necessary barrier protection for the wound, and may accelerate the healing process. Therefore, type 21 collagen not only shows 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 treating wounds.

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

[0215] Promote cell migration: ColpepA1 21, especially at a higher concentration (50ppm), showed a significant ability to accelerate healing through the HaCaT cell scratch test, which is crucial for rapid wound closure and shortening the healing cycle. At the dermal callus level, ColpepA121 can effectively promote the key cell migration link in the wound repair process.

[0216] Enhanced hydration: ColpepA1 21 significantly increased the expression of AQP3, indicating its importance in maintaining the hydration environment of the wound and surrounding tissues, and promoting normal cell function during wound healing, thereby strengthening the hydration capacity of skin cells. In wound repair, the enhanced expression of AQP3 helps maintain appropriate moisture in the wound area, thereby promoting normal cell 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 emphasized 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 for ColpepA1 21 in wound repair, especially as a key active ingredient in a novel composite collagen material. 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, making it an ideal candidate for developing highly effective wound repair treatment strategies and skin regeneration products. Therefore, ColpepA1 21 is expected to become a key factor in advancing the science of skin health and repair 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 at pH 5.5 at room temperature. Slowly add hyaluronic acid to the collagen solution and stir continuously to ensure uniform mixing. Slowly add EDC as a crosslinking agent to initiate the crosslinking reaction and 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, and then the mold was moved to 4°C and refrigerated 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 cross-linking agent 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, avoiding 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 example aims to evaluate the effect of type 21 collagen hydrogel dressing on barrier keratinocyte activity under cell culture conditions, especially its protective effect under oxidative stress. By comparing with type III collagen hydrogel dressing, the potential of type 21 collagen in promoting wound healing and cell regeneration is 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 cells / ml and 100 μl / well of the cell dilution was inoculated 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: only containing 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 by the method of Comparative Example 1 with an equal amount of 1000 ppm of type III collagen added;

[0246] Sample group C: type 21 collagen mixed hydrogel prepared by the method of Example 4 with an equal amount of 1000 ppm ColpepA1 21 added;

[0247] The activity test experiment was set up with a zero adjustment group, a normal group, a H2O2 modeling group and a sample group, and 3 replicate wells were set up in each group.

[0248] Liquid preparation:

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

[0250] CCK-8 working solution: Add 1 ml of CCK-8 mother 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% to 70%, the cell culture medium in each group was aspirated, the cells were washed once with PBS, then discarded and H2O2 working solution was added.

[0252] Sampling: After induction, the plates were washed twice with PBS, and 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; there was no cell inoculation in the zero-adjustment group, and only 100 μl of cell culture medium was added. After sampling, the 96-well plate was placed in an incubator (37°C, 5% CO2, 95% RH) and cultured for 24±2 h.

[0253] Cell viability detection: After culturing cells for 24±2h, first observe the cell status under a microscope, then discard the supernatant, add 100μl CCK-8 working solution to each well of each group, incubate at 37℃ in the dark for 2±0.5h, read the OD value at 450nm after incubation, and calculate the cell activity percentage 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 is used as the baseline, indicating the cell survival state in the 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 mitigation 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 shows 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 antioxidant and repair capabilities of cells. 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 for promoting wound healing, reducing inflammatory responses, and accelerating skin regeneration.

[0257] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention are all within the scope of the present invention.

Claims

1. A method for preparing a collagen material for wound repair, wherein: The method steps include: (1) Mixing collagen with auxiliary ingredients; (2) The collagen material solidifies and forms; (3) sterilizing the collagen material; 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 mixing is carried out at room temperature or under heating; and / or The solidification molding is carried out at room temperature or low temperature; and / or The sterilization includes any one of heat sterilization, chemical sterilization or radiation sterilization; Preferably, the sterilization is gamma ray sterilization or plasma sterilization.

3. The method according to claim 1 or 2, wherein: The method steps are selected from any one of the following methods (1)-(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; Low temperature curing, plasma sterilization; or Method (2): pre-soak collagen and auxiliary components in water and heat to dissolve; add hyaluronic acid solution, stir and solidify at room temperature; Low temperature curing, plasma sterilization; or Method (3): Mixing collagen and auxiliary components, spin coating on a substrate; drying and peeling, and gamma irradiation sterilization; or Method (4): Mix collagen and auxiliary ingredients, pour into a mold, freeze and then freeze-dry; Gas cross-linking, gamma ray sterilization.

4. The method according to any one of claims 1 to 3, wherein: The auxiliary ingredients are selected from one or more of the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, etc.

5. The method according to any one of claims 1 to 4, wherein: The concentration of the collagen is 0.0001% to 50% based on the total weight of the collagen material; Preferably, the concentration of the collagen is 1% to 20%.

6. The method according to any one of claims 1 to 5, wherein: The weight ratio of the collagen and the auxiliary ingredients is selected from: Collagen and antimicrobial agent: 5:1 to 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 to 50:1; or Collagen and emulsifier or thickener: one or more combinations in a ratio of 5:1 to 20:

1.

7. The method according to any one of claims 1 to 6, wherein: The method steps are selected from any one of the following methods (1)-(4): Method (1): a mixed material composed of type 21 collagen, hyaluronic acid and a cross-linking agent is subjected to cross-linking reaction in a sodium acetate buffer at room temperature; the mixed solution 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-soaking a mixed material of gelatin and collagen in water and heating to dissolve; adding hyaluronic acid solution, stirring and solidifying at room temperature; curing at low temperature, and plasma sterilization; or Method (3): Mix type 21 collagen and PLGA in a certain proportion, spin-coat the mixture on a substrate, dry and peel off, and sterilize with gamma rays; or Method (4): Type 21 collagen and chitosan are mixed in a certain proportion, poured into a mold, frozen, and then freeze-dried; Gas cross-linking, gamma ray sterilization; Preferably, 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; more preferably, the ratio of type 21 collagen is 3% (w / v); Preferably, in the method (1), the cross-linking agent is EDC; Preferably, the ratio in method (2) is: 2% (w / v) type 21 collagen solution, 1% (w / v) hyaluronic acid, and 3% (w / v) gelatin; Preferably, the ratio in the method (3) is: 1.5% (w / v) type 21 collagen solution, 2% (w / v) PLGA; Preferably, the ratio in method (4) is: 2.5% (w / v) type 21 collagen solution and 1.5% (w / v) chitosan.

8. A collagen material prepared according to the method according to any one of claims 1 to 7, wherein: The collagen material is used for repairing wounds after skin damage.

9. A medical composition, wherein: The composition comprises the collagen material prepared by the method of any one of claims 1 to 7 or the collagen material of claim 8; Preferably, the composition is a solid, liquid, semisolid or gel preparation; Preferably, 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; More preferably, 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, etc.; More preferably, the composition is in the form of a hydrogel dressing, more preferably a collagen-hyaluronic acid hydrogel dressing.

10. 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; preferably, the medical composition comprises the collagen material prepared by the method of any one of claims 1 to 7 or as described in claim 8.

Images