CN118949130A - Collagen material for facial filling - Google Patents

Abstract

The invention relates to the field of biological materials, in particular to a collagen material containing 21 type recombinant humanized collagen (ColpepA < 21 >) and application thereof in facial filling. The invention provides an effective solution for skin aging phenomenon by utilizing collagen, and also provides a collagen material for facial filling, which can rapidly realize the smoothing and tightening effects of facial skin by means of local injection or external coating, and can also promote the overall health condition of the skin after long-term use by promoting the natural synthesis of collagen in the skin, optimizing the hydration state and barrier function of the skin, enhancing the self-repairing capability of the skin and the like.

Description

Collagen material for facial filling

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a collagen material and application thereof in facial filling.

Background

Collagen, which is the most abundant protein in the human body, not only constitutes more than 30% of the total protein in the human body, but also is a key element for supporting various tissue structures and functions of the human body. It is widely distributed in various connective tissues such as skin, bones, tendons, blood vessels and the like, constructs a three-dimensional network structure and provides necessary mechanical strength and stability for the tissues. Collagen plays a vital role in skin health, and is responsible for maintaining skin firmness, elasticity, and maintaining smoothness and luster of the skin surface. However, with age, the natural rate of collagen production gradually slows down, together with the effects of ultraviolet radiation, environmental pollution and bad lifestyle, resulting in progressive degradation and damage of existing collagen, and the skin begins to develop signs of aging such as sagging, fine lines and wrinkles. This not only affects the appearance of the skin, but may also negatively affect bone density, tendon elasticity and vascular stability, and thus the overall health.

In facial filling products, the application of collagen is aimed at directly supplementing the collagen needed by the skin to fill the loss of skin volume caused by collagen loss, restore the firmness and elasticity of the skin, and reduce wrinkles and fine lines, thereby achieving the effect of rejuvenation. Collagen is an ideal choice in facial filling and anti-aging treatment, taking advantage of its natural role and biocompatibility in skin structures. However, current collagen facial filling products face challenges of how to improve the stability of collagen, extend its persistence in the skin, and how to enhance its permeability and bioavailability. In addition, the precise matching and supplementation of different types of collagen requirements during aging is also an important point in the need of further optimization and improvement of the existing products. Current scientific research and product development are working to address these issues, through innovative technology and formulation improvements, to achieve more effective, longer lasting collagen supplementation and anti-aging effects.

In this context, intensive research into specific types of collagen, such as type 21 collagen (COL 21 A1), is particularly important. Type 21 collagen is a non-fibrous collagen, which is relatively rare in adults and accounts for less than 1% of the total collagen. Type 21 collagen has been relatively less studied than other more well known collagen types. Type 21 collagen belongs to the family of small molecular collagens, and as part of the family of FACIT (fiber-related collagen with interrupted triple helices), type 21 collagen plays a vital role in the attachment of extracellular matrix components. Studies in Chou MY and Li HC have shown that it may play a key role in the assembly of extracellular matrix during angiogenesis [ Chou MY, li HC (March 2002) ]. In addition, the gene expression of type 21 collagen is regulated by developmental stages, and the expression level of fetal stages is high, suggesting that it may play an important role in the development of various tissues. Surendran et al, which disclose their function in heart and aortic collagen formation and in connection with vascular remodeling and hypertension, further point to their potential value in promoting skin health and aging [ Surendran et al (2016) ]. This provides a new perspective for the research of COL21A1 in promoting skin health and anti-aging.

Although collagen materials on the market rely primarily on traditional collagen or derivatives thereof to supplement collagen loss in the skin, the effectiveness of these traditional products is often limited by the size of the collagen molecules and the absorption rate of the skin, and they are difficult to optimize for the unique biological functions of a particular type of collagen. However, based on the unique biological properties and mechanism of action of type 21 collagen, the developed facial filling products are expected to surpass traditional solutions, providing anti-aging therapies that are more targeted, highly bioavailable and have multiple efficacy. These products not only improve the appearance of the skin, but also promote overall health and delay the progression of aging through their effects on extracellular matrix composition and vascular health, representing an important direction for the future development of facial filling technology.

Disclosure of Invention

The invention relates to a collagen material and application thereof in facial filling.

In one aspect, the invention provides a type 21 recombinant humanized collagen, wherein the amino acid sequence of the collagen is selected from the group consisting of SEQ ID No. 1:

GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG

The collagen of the specific amino acid sequence is optimally designed to enhance its bioavailability and efficacy in the skin.

In one embodiment, the collagen of the present invention is capable of enhancing skin moisture regulation and optimizing barrier function.

In one embodiment, the collagen of the present invention is capable of promoting the expression of aquaporin AQP3 to achieve the effects of promoting overall health and anti-aging of the skin by increasing skin water retention and decreasing water evaporation rate, improving skin dryness and tightness.

In one embodiment, the collagen of the present invention is capable of reducing skin fine lines and wrinkles, increasing skin firmness, and providing critical support for anti-aging and skin rejuvenation.

In one embodiment, the collagen of the present invention is capable of promoting the formation of type III collagen, and significantly increasing the amount of type III collagen, which can enhance skin elasticity and repair ability, and particularly shows significant benefits in promoting wound healing and combating skin aging.

In one embodiment, the collagen of the present invention enhances the structural integrity of the skin by promoting the production of type I and type III collagen, enhancing elasticity, thereby reducing signs of aging and promoting skin health.

In one embodiment, the collagen of the present invention demonstrates potential as an active ingredient in a facial filling product in an ex vivo skin model after UVA and UVB irradiation.

In one embodiment, the collagen of the present invention exhibits protective and repair effects on skin lesions.

In one embodiment, the collagen of the present invention contributes to an increase in the thickness of the viable cell layer of the epidermis.

In one embodiment, the collagen of the present invention contributes to the enhancement of collagen fiber density.

In one embodiment, the collagen of the present invention contributes to the enhancement of type I collagen content.

In one embodiment, the collagen of the present invention contributes to the enhancement of type III collagen content.

In one embodiment, the collagen of the present invention contributes to the elevation of type IV collagen content.

In one embodiment, the collagen according to the invention contributes to an increase in the XVII type collagen content.

Another aspect of the present invention provides a collagen material for facial filling, wherein the collagen material for facial filling comprises recombinant humanized collagen type 21 (ColpepA 1) having an amino acid sequence selected from SEQ ID No.1, and further comprising an auxiliary component selected from one or more of the following: filling auxiliary agents, solvents, antioxidants, emulsifiers, moisturizers, stabilizers, pH regulators, thickeners, and the like;

preferably, the filling aid comprises hyaluronic acid, calcium hydroxyapatite, polylactic acid, platelet rich plasma, polymethyl methacrylate microspheres, liquid injectable silica gel, or the like.

In an embodiment, the collagen material of the present invention, wherein the concentration of the collagen is 0.0001% -50% based on the total weight of the collagen material.

In an 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:

in one embodiment, the ratio of collagen to antimicrobial agent is selected from 10:1 to 30:1; suitable antimicrobial concentrations are effective in inhibiting bacterial growth, but too high may cause skin irritation and allergic reactions.

In one embodiment, the ratio of collagen to anti-inflammatory agent is selected from 15:1 to 100:1; the facial filling process may cause minor inflammation, so proper use of anti-inflammatory agents helps to alleviate these symptoms, but the amount thereof needs to be controlled to avoid inhibiting normal skin function.

In one embodiment, the collagen to growth factor ratio is selected from 100:1 to 300:1; growth factors are very beneficial for promoting collagen synthesis and natural skin recovery processes, but should be carefully used in facial filling to avoid overgrowth or uneven skin texture.

In one embodiment, the collagen to humectant ratio is selected from 1:1 to 8:1; moisturizers are particularly important in facial filling because they help maintain the smooth and full feel of the skin. Increasing the proportion of the humectant can effectively improve the durability and natural feel of the filling effect.

In one embodiment, the collagen to antioxidant ratio is selected from 10:1 to 50:1; antioxidants can prevent oxidation of the formulation but should be used in amounts controlled so as not to affect the long-term stability of the product.

In one embodiment, the ratio of collagen to emulsifier or thickener is selected from 5:1 to 20:1; which helps to improve the texture and stability of the product, but should be used in an amount to ensure the suitability and comfort of the final product.

In another aspect of the invention, a medical composition is provided, wherein the composition comprises recombinant humanized collagen type 21, the amino acid sequence of which is selected from SEQ ID No.1 or any of the foregoing collagen materials.

In one embodiment, the formulation forms of the medical composition of the present invention include, but are not limited to, the following forms:

injectable formulations, preferably including but not limited to injectable hydrogels, compound injectables, and the like, are used to provide deep facial filling effects by surgical direct injection into the skin.

Topical application formulations, preferably including but not limited to, application gels, creams or lotions, are suitable for daily skin care, helping to reduce wrinkles and restore skin elasticity.

Solution formulations, preferably including but not limited to collagen solutions for microneedles or microneedle rollers, promote collagen distribution and absorption in the skin by minimally invasive means.

In one embodiment, the product of the invention may further be supplemented with a pharmaceutically, food, nutraceutical or dietary acceptable carrier. For example, a variety of compatible solid or liquid fillers, gel materials or solvents may be employed, and these carriers should be suitable for human use, of sufficient purity and low toxicity. Common carriers include cellulose and its derivatives, polyols such as glycerol, and other pharmaceutical excipients such as talc, magnesium stearate, and the like.

In another aspect, the invention provides an application of the type 21 recombinant humanized collagen in preparing a medical composition for facial filling, wherein the amino acid sequence of the collagen comprises the amino acid sequence shown in SEQ ID No. 1.

In another aspect, the invention provides the use of any of the foregoing collagen materials for the preparation of a medical composition for facial filling.

Another aspect of the present invention provides a method for preparing the collagen material for facial filling.

In one embodiment, the collagen material is a collagen composite injection, wherein the raw materials used are 2% -10% (w/v) of type 21 collagen and 0.1% -1% (w/v) of hyaluronic acid; vitamin C0.05% (w/v) was used as an antioxidant, and physiological saline was used as a solvent; the preparation process comprises the following steps:

Dissolving an antioxidant;

Preparing a collagen solution;

adding hyaluronic acid;

mixing an antioxidant solution;

pH and viscosity adjustment;

sterile filtration and filling.

In one embodiment, the collagen material of the present invention is a collagen/hyaluronic acid blended injection hydrogel; wherein the hydrogel structure is reinforced by using a cross-linking agent EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), and the optimal formulation is determined by adjusting the mixture of collagen and hyaluronic acid in different proportions and their reaction with EDC in different concentrations; the raw materials are 1% -5% (w/v) of type 21 collagen and 0.5% -2% (w/v) of hyaluronic acid; the formula also comprises phosphate buffer solution as a dissolution base, and glycerin 1% -5% (w/v) is added as a humectant.

In one embodiment, the collagen material of the present invention is a collagen solution for a microneedle/microneedle roller, wherein the collagen solution is suitable for use in a microneedle or microneedle roller, and the distribution and absorption of collagen in skin is promoted by a minimally invasive manner. The collagen concentration in the formulation is higher, 3% -10% (w/v), to enhance its absorption through the microneedle channels. The auxiliary materials comprise 0.1% -1% (w/v) of hyaluronic acid to enhance moisturizing effect, 0.5% -2% (w/v) of urea as a penetration enhancer to assist collagen to penetrate skin, and buffer solution with pH value (pH 5.5-7.0) close to that of skin.

Advantages of the invention

Type 1.21 collagen promotes the formation of type I and type III collagen, which are key components of skin elasticity and structure. This capability provides a new mechanism for collagen-based skin anti-aging strategies. By promoting the production of type I and type III collagen, type 21 collagen not only accelerates the healing process of the wound, but also improves the quality of the skin after repair. This is particularly important for facial filling, as high quality skin repair results can provide a more natural, durable cosmetic effect, reduce scar formation, and improve skin elasticity and structural stability. Meanwhile, the type 21 collagen achieves the mechanism of reducing fine lines and wrinkles and improving skin firmness and elasticity by increasing the generation of type I and type III collagen, which means that a non-invasive skin rejuvenation solution, such as a microneedle roller/microneedle therapy/external product is provided in the plastic and cosmetic industries, and the collagen is delivered to deeper layers of the skin/the generation of type I and type III collagen is promoted without injection, so that the filling effect is achieved.

2. Since type 21 collagen naturally exists in the human body, it is particularly excellent in biocompatibility and low immunogenicity when used as a facial filling material. This reduces the risk of an immune response after injection, providing a safer treatment option for the patient.

3. In addition to being an effective facial filling material, type 21 collagen has a variety of potential additional values for promoting skin health, such as anti-aging and improving skin hydration. This makes it possible not only to use it as a filling material, but also as an active ingredient for skin care and cosmetic products, providing a more comprehensive cosmetic solution. The recombinant humanized collagen type 21 (ColpepA 1 21) of the present invention exhibits significant advantages in terms of skin aging resistance. The unique efficacy and optimized molecular characteristics of ColpepA 1.21, while providing basic collagen supplementation, make it a distinct feature in numerous collagen materials.

Drawings

FIG. 1 shows the relative fluorescence intensity of type III collagen in each cell group tested.

Fig. 2 shows a bar graph of the results of the thickness of the epidermal living cell layer, wherein # represents the significant difference (P < 0.01) compared to the blank control group, and # represents the significant difference (P < 0.01) compared to the negative control group, as in fig. 3 to 7.

Fig. 3 shows a histogram of collagen fiber relative area results.

FIG. 4 shows a histogram of Collagen I relative Integrated Optical Density (IOD) values.

FIG. 5 shows a histogram of Collagen III relative Integrated Optical Density (IOD) values.

Fig. 6 shows a histogram of Collagen IV versus Integrated Optical Density (IOD)/Area values.

FIG. 7 shows a histogram of the Integrated Optical Density (IOD)/Area values of Collagen XVII.

Fig. 8 shows the effect of ColpepA 1.1.21 compound injection on cellular HYP content under oxidative stress.

Correlation definition

Unless specifically indicated, the following terms used in the specification and claims have the following meanings:

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, bone, tendon, ligament, cornea, and the like. The main functions of these proteins are to provide body tissue strength, stability and elasticity, ensuring proper performance of various physiological functions. There are at least 28 types of collagen known today, each of which plays a unique role and function in the body, creating a complex and finely tuned biological system.

As used herein, the term "type 21 collagen (COL 21 A1)" refers to a type of collagen that exists in the human body and belongs to the family of small molecular collagens. As part of the family of facility (fiber-related collagen with interrupted triple helices), type 21 collagen plays a critical role in the attachment of extracellular matrix components. This collagen promotes interactions and links between different collagen types through its unique molecular structure, supporting the integrity, ordering, and function of extracellular matrix 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 the skin repair mechanisms. Although type 21 collagen is relatively rare in the human adult, accounting for less than 1% of the total amount of collagen, its potential roles in cellular processes such as migration, proliferation and differentiation, as well as in maintaining extracellular matrix structure and promoting tissue regeneration, are important directions for future research.

As used herein, the term "recombinant humanized collagen type 21 (ColpepA 1) refers to a protein that mimics type 21 collagen naturally occurring in humans, expressed in pichia pastoris by genetic recombination techniques. The protein has biological function and structural characteristics similar to natural type 21 collagen, and the amino acid sequence of the protein has specific codes, so as to improve the health and anti-aging capability of skin.

As used herein, the term "HaCaT (human keratinocyte)" refers to an immortalized human keratinocyte cell line, which is widely used in research into skin biology, skin pathology, and drug toxicity tests, etc., and is considered as a valuable research tool because it is capable of mimicking the behavior of normal epidermal cells.

As used herein, the term "human skin fibroblast HFF (Human Foreskin Fibroblasts, human foreskin fibroblast)" refers to fibroblasts isolated from human fetal foreskin, which are important components of skin structure responsible for the synthesis of collagen and other extracellular matrix proteins, commonly used for the study of skin aging, wound healing, collagen synthesis, and the like.

As used herein, the term "human skin fibroblast HDF (Human Dermal Fibroblasts, human dermal fibroblast)" refers to a fibroblast isolated from the human dermis. These cells play a central role in the formation of skin structures, mainly responsible for the synthesis of collagen and other extracellular matrix proteins. HDF cells are commonly used in the fields of research of skin aging, wound healing, disease models, drug screening, and the like.

As used herein, the term "composition" refers to a formulation containing recombinant humanized collagen type 21 and possibly including other auxiliary ingredients such as moisturizers, antioxidants, or whitening ingredients that work together to provide a more comprehensive skin care effect.

As used herein, the term "tgfβ1" (transforming growth factor β1) refers to a multifunctional cytokine capable of modulating cell proliferation, differentiation and migration. In skin physiology, tgfβ1 has important effects on collagen synthesis, wound healing processes and anti-inflammatory effects.

As used herein, the term "HPR (oxalophenolate)" is a synthetic retinol derivative, commonly used in skin care products, having the ability to promote cell renewal, enhance skin repair, and improve the state of skin aging.

As used herein, the term "ex vivo skin model" refers to a skin tissue sample extracted from a living body and maintained under laboratory conditions to simulate and study the biological properties of skin in vitro and the response to various treatments.

As used herein, the terms "UVA and UVB radiation" refer to two main types of ultraviolet radiation. UVA radiation (long wave ultraviolet) can penetrate into the dermis of the skin, resulting in long-term damage to the skin such as skin aging. UVB radiation (medium wave ultraviolet) affects mainly the epidermis layer of the skin and is a major cause of sunburn.

As used herein, the term "tissue morphology" refers to the microstructural state of the skin, including the shape, size, arrangement and interaction of cells, as observed by staining and microscopy. Changes in tissue morphology, such as changes in epidermis thickness and dermis density, can intuitively reflect skin response to external factors, including damage from ultraviolet radiation, and the effectiveness of therapeutic and repair measures.

As used herein, the term "collagen fibers" refers to the fibrous structure of the dermis layer of the skin consisting essentially of type I and type III collagen. Collagen fibers provide the necessary elasticity and firmness to the skin and affect its overall appearance and feel. The change in collagen fiber content and quality is a key indicator for measuring the ability of facial filling products to strengthen skin support structures, improve skin sagging, and reduce wrinkles.

As used herein, the term "type I Collagen" (Collagen I) refers to the most abundant type of Collagen in the human body, which is the fundamental constituent molecule that makes up many tissues such as skin, bone and tendons. Its main function is to provide structural support and physical strength to these tissues, maintaining skin elasticity and firmness. The naturally occurring decrease in type I collagen is closely related to skin laxity and wrinkle formation with age. In facial filling products, the increase in type I collagen is considered an important indicator of product effectiveness, particularly in terms of anti-aging and improving skin structure.

As used herein, the term "type III Collagen" (Collagen III) refers to a type of Collagen that is present in the skin structure together with type I Collagen, especially in young skin. It plays a critical role in maintaining skin elasticity and assisting in the healing process of damaged skin. The temporary increase in type III collagen helps repair and rebuild the structure of the skin after skin injury. The decrease in its content with age may affect the elasticity and repairability of the skin. The increased type III collagen content in the face pack product shows the potential of the product to promote natural skin repair and enhance skin elasticity.

As used herein, the term "Collagen IV" (Collagen IV) refers to the major component of the basement membrane, a special layer located between the epidermis and dermis that plays a key role in skin structural stability and barrier function. Type IV collagen forms the network structure of the basement membrane, providing a support layer for skin cells to maintain physical stability and promote the repair process. With age or damage to the external environment, such as ultraviolet radiation, the function of the basement membrane may be impaired, leading to skin aging. Thus, an increase in type IV collagen content in the facial filling product can help restore and protect the basement membrane of the skin, helping to promote overall health and appearance of the skin.

As used herein, the term "Collagen type XVII" (Collagen XVII) refers to a transmembrane Collagen that is primarily localized on the epidermal basal cell membrane of the skin and participates in maintaining cell adhesion and the barrier function of the skin by interacting with other components in the basal membrane. It plays a critical role in maintaining skin integrity and responding to external pressure. Damaged or aged skin may exhibit reduced expression of collagen type XVII, resulting in reduced skin barrier function and skin laxity. Thus, increasing the level of XVII type collagen in a facial filling product can help to enhance the skin's ability to self-repair and barrier function, reducing the damaging effects of external environmental factors on the skin.

As used herein, the term "composition" or "formulation" refers to a mixture comprising one or more of the collagen of the present application and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

As used herein, the term "antioxidant" refers to a class of substances capable of neutralizing free radicals, slowing or preventing cellular damage, which has an important role in reducing oxidative stress caused by environmental factors and preventing skin aging.

As used herein, the term "emulsifier" refers to a substance that can aid in the mixing of oil and water to form a stable emulsion, which is used in skin care products to enhance the texture and stability of the product.

As used herein, the term "moisturizer" refers to a substance that is capable of attracting or retaining moisture, helping the skin retain moisture, and has a critical effect on maintaining the hydration state of the skin and preventing dryness.

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 salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opines), alanines, octopine, glycine (strombine)) and the N-oxides of Trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α -casein, globulin, α -lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, and the like, may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of polyhydric alcohols, amino acids, salts and saccharides. The preferred salts are sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or salts thereof (e.g., arginine hydrochloride), glycine, 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, arginine hydrochloride-sucrose.

As used herein, the term "Hydroxyproline (HYP)" refers to a non-essential amino acid, which is mainly present in collagen and is an important component of the tripeptide sequence of collagen. HYP plays a key role in the stable structure and tension resistance of collagen and is commonly used as a biomarker for evaluating collagen content and synthetic activity.

As used herein, the term "H ₂O₂ oxidative stress treatment" refers to an experimental technique that uses hydrogen peroxide (H ₂O₂) to treat cells that mimics the oxidative stress experienced by cells under physiological or pathological conditions. This treatment is often used to study the response mechanism of cells to oxidative damage, protection strategies, and viability under oxidative stress, particularly in studies discussing anti-aging, antioxidant, and disease models.

Detailed Description

In particular embodiments, the materials and methods used are as follows:

cell lines: haCaT (human keratinocyte) and HFF-1 (human fibroblast) were purchased from the afterglow organism.

Reagent: CCK-8 kit (Japan Tonic CK 04) was used for cell activity evaluation, AQP3 (Shanghai enzyme-exemption) and type I collagen kit (Shanghai enzyme-exemption CMM2023H 1) was used for related protein expression analysis.

Example 1: III collagen production

This example is directed to evaluating the effect of type 21 recombinant humanized collagen (ColpepA 1) on stimulating type III collagen production. Type III collagen is a key component for ensuring the structural stability and elasticity of the skin, and has important significance for the younger and damage recovery of facial skin. Through this study we aimed to reveal the potential of ColpepA1 as an active ingredient in facial filling and anti-aging collagen materials.

Experimental grouping:

blank Control (Control): no treatment was performed to assess the level of type iii collagen expression of the cells in the intact state.

Negative control group: cells were treated with 5J/cm UVA damage to mimic the skin damage effect in sunlight.

Positive control group (HPR): 0.001% HPR (oxalic acid phenolphthalein) was added after UVA injury, a therapeutic agent known to aid in skin repair.

ColpepA1 experimental group 21: 10 ppm and 50 ppm of type 21 recombinant humanized collagen were added after UVA injury, respectively.

Culturing fibroblast HFF-1 respectively, plating and culturing 24h, adding a sample for treatment after UVA induction when the cell fusion degree is 70-80%, culturing 24h, and quantitatively analyzing the relative fluorescence intensity of the III type collagen in the cells of each experimental group by using an Immunofluorescence (IF) detection method to detect the expression of the III type collagen.

Data collection and analysis:

FIG. 1 shows the relative fluorescence intensity results of type III collagen obtained by immunofluorescence techniques. As can be seen from the graph, the blank group showed normal level of fluorescence intensity of type iii collagen, which is the baseline expression level. The type iii collagen fluorescence intensity of the UVA negative control group was significantly lower than that of the control group, showing extracellular matrix damage caused by UVA irradiation. The positive control group with 0.001% HPR was increased relative to the UVA negative control group, but still lower than the blank control group, indicating that HPR had some extracellular matrix protection and repair. The ColpepA1 experiment group, after 50 ppm ColpepA1 21 is added, has obviously increased fluorescence intensity of III type collagen, and compared with the UVA negative control group, the promotion rate is 25.42%, which shows that ColpepA1 has obvious III type collagen generation promotion effect.

The effect of the 21-type recombinant humanized collagen on promoting III-type collagen generation obviously improves the repair capability after skin damage and increases the elasticity of the skin, which is very important for the anti-aging field. The effect not only supports the application of ColpepA <1 > 21 in collagen materials for facial filling, but also emphasizes the remarkable potential of the collagen materials in promoting the repair of damaged skin, improving the skin elasticity and resisting aging. The collagen material for facial filling provides precious scientific basis, shows the unique value of ColpepA < 21 > in improving skin health and delaying aging process, and lays a solid foundation for further development and commercial application of the collagen material for aging resistance.

Example 2: ex vivo skin model test

This experiment was intended to evaluate the effect of ColpepA 1.21 on improving skin elasticity and reducing wrinkles, in particular skin repair and protection after exposure to UVA and UVB radiation. The potential of ColpepA a 21 for use in facial filling products was evaluated by simulating uv exposure in a daily setting. The experiment is to analyze the change of the isolated skin tissue after ultraviolet irradiation, detect the change of the tissue morphology, collagen fiber, type I collagen, type III collagen, type IV collagen and type XVII collagen content, evaluate the influence on the skin structure and the anti-aging potential thereof, and analyze the application effect of type 21 collagen in the aspect of facial filling.

Experimental grouping:

Blank control group: no treatment was performed.

Negative control group: no specific treatment is illustrated, but generally refers to a group to which no sample to be tested is added, which is subjected only to the same external conditions.

Positive control group: as an effect control, a mixture of VC (vitamin C) and VE (vitamin E) known to have a tightening and anti-wrinkle effect was used.

Sample group (collagen type 21 group, colpepA 1) of: treatment with recombinant humanized collagen type 21 (ColpepA 1) at a concentration of 0.0125% (v/v) was performed in order to test its tightening and anti-wrinkle effects.

The main reagent comprises: isolated skin tissue culture (Guangdong Boxi organism), PBS (Soy pal), vitamin C (VC, sigma), vitamin E (VE, sigma), absolute ethanol (national drug), xylene (national drug), hematoxylin (Biyun), eosin (Biyun), collagen I antibody (Proteintech), collagen III antibody (Abcam), collagen IV antibody (Abcam), collagen XVII antibody (Abcam), paraformaldehyde (Biosharp).

The main equipment comprises: CO ₂ incubator (Thermo, 1501), ultra clean bench (sulbactam, SW-CJ-1F), overhead microscope (Olympus, BX 53), UVA irradiation instrument (philips), UVB irradiation instrument (philips), fluorescence microscope (Leica, DM 2500).

1. Tissue treatment

Freshly obtained skin tissue was immersed in 75% alcohol for 30 seconds to remove surface impurities and microorganisms. Three washes with sterile PBS buffer were used to further clean and prepare the tissue. The skin was cut into 24±2 mm pieces of tissue with the epidermis facing up and the dermis facing down and placed in a culture mold. The culture mold was placed in 6-well plates, 3.7 mL culture medium was added to each well, and the culture was carried out in an incubator at 37℃with 5% CO ₂, with the culture medium being changed every day.

2. Irradiation and administration

After 2 days of incubation, irradiation and administration of the ex vivo skin tissue was started according to the test packets and their corresponding treatment conditions. The irradiation dose was set to UVA (30J/cm. Sup. Nd) and UVB (50J/cm. Sup. Nd) and irradiation was continued for 4 days. Fresh culture medium was changed after each irradiation and the dosing treatment was performed. The positive control group adopts a submerged administration mode; the sample group adopts a surface administration mode. After irradiation and administration for 4 days, the isolated skin tissue was cultured for 3 days, during which time no irradiation was performed and only sample administration was performed.

3. Cleaning

After the incubation, the residual reagent on the surface of the model was washed with sterile PBS solution, and the residual liquid inside and outside the model was gently wiped off with a sterile cotton swab.

4. Tissue morphology and collagen fiber detection

After washing, skin tissue was fixed with 4% paraformaldehyde, tissue embedded and sectioned, H & E stained and Masson stained, respectively, photographed using a microscope, and analyzed using Image-Pro Plus Image processing software.

5. Immunofluorescence detection

Taking a model for detection, fixing with 4% paraformaldehyde for 24 hours, performing immunofluorescence detection, photographing under a microscope for observation, and collecting and analyzing pictures.

6. Lifting rate calculation

According to the immunofluorescence result, the improvement rate of each index of each experimental group is calculated by using the following formula, and the effect of the type 21 collagen is further evaluated.

Improvement ratio (%) = (experimental group-blank control group)/blank control group×100%

7. Statistical analysis of results

The plots were plotted using GRAPHPAD PRISM software and the results expressed as mean ± standard deviation, and the differences between the groups were compared by t-test statistical analysis.

Example 3-1: tissue morphology testing

The tissue morphology reflects the microstructure state of the skin and is an important index for evaluating skin health and aging degree. In the evaluation of the face pack product, the effectiveness of the product in resisting skin damage, promoting skin repair, improving skin firmness and the like can be intuitively understood by observing the changes of the epidermis thickness, dermis density and the like of the skin after treatment. When the skin is affected by external factors (such as UV radiation), its tissue structure changes, such as thinning of the epidermis layer, reduction of collagen fibers in the dermis layer, etc., which are all manifestations of skin aging. An effective facial filling product should promote proliferation of skin cells, restore or increase epidermal thickness, and enhance the density and elasticity of dermis layers, thereby improving the appearance and texture of skin.

Fig. 2 shows a bar graph of the results of the thickness of the epidermal living cell layer, in which # indicates significant difference (P < 0.01) compared to the blank control group, and # indicates significant difference (P < 0.01) compared to the negative control group. As shown, the Blank (BC) set demonstrates a baseline level of epidermal viable cell layer thickness without any treatment. The negative control group (NC) showed a significant decrease in the thickness of the epidermal living cell layer of this group after UV irradiation, indicating that UV irradiation caused significant damage to the skin structure, resulting in thinning of the epidermal layer. After the positive control group (PC) is treated by known anti-aging components (such as VC+VE), the thickness of the epidermis of the skin is increased, and the positive control substance has a certain effect of resisting UV radiation damage. The sample group (collagen type 21 ColpepA1, 0.0125%) showed a significant increase in the thickness of the epidermal living cell layer (30.52% increase rate) compared to the negative control group, indicating that ColpepA 121 significantly promoted an increase in the thickness of the epidermis against damage caused by UV irradiation.

The results indicate that ColpepA a 21 provides an effective protection mechanism against damage from UV radiation, increases skin elasticity and firmness, which means that it is effective in improving skin structure, restoring or maintaining the youthful state of the skin for a facial filling product, with potential therapeutic and cosmetic benefits.

Example 3-2: collagen fiber test

Collagen fibers are the main component of the dermis layer of skin, mainly composed of type I and type III collagen, and have a decisive influence on the elasticity, firmness and overall appearance of skin. In facial filling product assessment, the ability of the product to strengthen skin support structures, improve skin sagging, and reduce wrinkles can be assessed by measuring the content and quality of collagen fibers. As the skin ages or is affected by external factors such as ultraviolet rays, collagen fibers in the skin gradually decrease and become damaged, resulting in loss of elasticity and firmness of the skin, and wrinkles and sagging. The facial filling product can help restore skin elasticity and firmness by stimulating collagen production, and reduce wrinkles.

Fig. 3 shows a histogram of collagen fiber relative area results. As shown, the relative area of collagen fibers can be considered as a reference value for normal levels without any treatment in the Blank (BC). The Negative Control (NC) showed a significant decrease in the relative area of collagen fibers under UV irradiation, indicating that UV irradiation resulted in collagen fiber damage and degradation of skin structure. The positive control group (PC) was treated with skin samples known to have anti-aging effect, and the relative areas of collagen fibers were restored, indicating that the positive control substance could combat the damage of collagen fibers caused by UV irradiation. Compared with the negative control group, the sample group (type 21 collagen ColpepA1, 0.0125%) has obviously increased relative area of collagen fiber and the lifting rate is 43.48%. This means ColpepA that ColpepA a 21 can significantly promote the increase of collagen fibers, improve the firmness and elasticity of the skin, and combat the phenomenon of aging.

The results show that ColpepA a 21 as an active ingredient, which has a remarkable skin tightening and anti-aging effect for facial filling products, is demonstrated in the increase of collagen fiber content, making it a valuable ingredient.

Examples 3-3: type I Collagen (Collagen I) test

Type I collagen is the most abundant type of collagen constituting human skin, accounting for about 80% of the total protein of the dermis layer of the skin, and its content is directly related to the structural integrity and function of the skin. With age, the production of type I collagen decreases and the skin becomes flaccid and wrinkled. Thus, promoting the production of type I collagen is a key goal in the development of anti-aging and skin repair products. In the assessment of facial filling products, the increase in type I collagen is considered an important indicator of improving skin structure, enhancing skin firmness and elasticity.

FIG. 4 shows a histogram of Collagen I relative Integrated Optical Density (IOD) values. As shown, the Blank (BC) group, without any treatment, shows the IOD value of Collagen I at baseline, providing a benchmark for subsequent comparisons. The Negative Control (NC) showed a significant decrease in the IOD value of Collagen I after UV irradiation, indicating that UV irradiation caused significant damage to Collagen I, resulting in a decrease in its content in the dermis layer. The significant increase in the IOD value of the Collagen I in the Positive Control (PC) compared to the negative control suggests that vc+ve treatment in the positive control helps to restore the Collagen I content and combat the damaging effects of UV. The sample group (Collagen type 21 ColpepA, 1, 0.0125%) showed that the IOD value of Collagen I was similar to that of the positive control group, and also significantly higher than that of the negative control group, with a rise of 159.38%. This indicates that Collagen type 21 is effective in promoting the increase in Collagen I, which is beneficial for strengthening the underlying structure of the skin and improving the firmness and elasticity of the skin.

The results show that ColpepA a 21 is an effective component, can significantly enhance the content of Collagen I in skin, and is very helpful for preventing and treating UV-induced skin injury, reducing wrinkle formation and improving skin firmness. This also provides a scientific basis for ColpepA's 21 application in face filling products.

Examples 3-4: type III Collagen (Collagen III) test

Type III collagen is another important collagen in the skin, especially in the skin of children and adolescents, and forms the main supporting structure of the skin together with type I collagen. It plays a vital role in skin elasticity and repair, especially in the healing process following skin injury, the production of type III collagen is temporarily increased, helping to repair damaged skin structures. With age, the proportion of type III collagen is gradually reduced relative to type I collagen, affecting the elasticity and repairability of the skin. The increase in type III collagen reflects the potential of the product in promoting skin repair and enhancing skin elasticity. For damaged skin or aged skin, the product capable of increasing the content of type III collagen is helpful for accelerating the natural repair process of the skin and improving the overall texture and appearance of the skin. The increase in type III collagen is seen as the ability of the facial filling product to promote skin regeneration and restore skin health.

FIG. 5 shows a histogram of Collagen III relative Integrated Optical Density (IOD) values. As shown, the Blank (BC) exhibited Collagen III IOD values for baseline levels. The IOD value of type III collagen was significantly reduced after UV irradiation of the negative control group (NC), indicating that UV irradiation significantly damaged type III collagen in the skin. Positive control group (PC): the type III Collagen content increased significantly after known anti-aging treatments, indicating that the positive control treatment helped restore the Collagen III content after UV irradiation damage. The IOD value of the sample group (type 21 collagen ColpepA1, 0.0125%) of type III collagen was significantly higher than that of the negative control group, with a lifting rate of 173.91%, which indicates that type 21 collagen significantly promoted the production of type III collagen, improved skin elasticity and reduced formation of wrinkles.

The results indicate that ColpepA a 21 can be an active ingredient of a face pack product, particularly in promoting skin elasticity and anti-aging. The experimental result emphasizes the remarkable influence of the skin texture and appearance on the increase of the III type collagen, and further shows that the skin texture and appearance can be improved, and a scientific basis is provided for the efficacy of the facial filling product.

Examples 3 to 5: type IV Collagen (Collagen IV) test

Type IV collagen forms the network of the basal membrane of the skin, providing a support layer to which the superficial cells of the skin can be firmly attached. The structure not only maintains the physical stability of the skin, but also plays a key role in the repair process of the skin, the transmission of nutrient substances and the maintenance of barrier functions. With aging or damage from the external environment (e.g., ultraviolet radiation), destruction of type IV collagen can result in impaired basement membrane function, thereby accelerating the skin aging process. Therefore, the effect of the facial filling product in increasing the type IV collagen content can help restore and protect the basement membrane of the skin, improving the overall health and appearance of the skin.

FIG. 6 shows a column of Collagen IV relative Integrated Optical Density (IOD)/Area values. As shown, the Blank (BC) represents baseline levels of Collagen IV in untreated skin samples. The Negative Control (NC) showed a significant decrease in the content of Collagen IV under the influence of UV irradiation, indicating that UV irradiation successfully induced conditions of skin aging and caused damage to the basement membrane. The significant increase in the content of Collagen IV in the Positive Control (PC) compared to the negative control (labeled as x), indicates that the treatment is effective against damage caused by UV irradiation by a component that promotes the synthesis of or slows down the degradation of Collagen IV. The cologen IV content of the sample group (Collagen type 21 ColpepA1, 0.0125%) was also significantly higher than that of the negative control group with a lifting rate of 102.78%, indicating that ColpepA1 significantly promoted the synthesis or stability of cologen IV, helping to restore the structure and function of the basement membrane.

The results indicate that the use of ColpepA a 21 shows positive effects in improving skin health and preventing premature aging, which provides a scientific basis for its use in facial filling and skin care products. ColpepA1 helps to maintain the youthful condition of the skin by enhancing the structural integrity and function of the basement membrane, a valuable component in anti-aging strategies.

Examples 3 to 6: collagen XVII (Collagen XVII) test

The XVII type collagen is a transmembrane type collagen, which is located on the cell membrane of epidermal basal cells, and which stabilizes cell attachment and assists in cell-to-cell signaling by interacting with other components in the basal membrane. This adhesion is critical to the integrity and barrier function of the skin. In the case of damaged or aged skin, the expression of type XVII collagen may be affected, resulting in reduced adhesion of epidermal cells and reduced skin barrier function. Thus, by external application of a facial filling product that increases the collagen content of XVII type, it can help strengthen the skin's barrier function and promote skin health. In the evaluation of facial filling products, an increase in type XVII collagen is believed to enhance the self-healing ability of the skin, reduce the damaging effects of external environmental factors, and combat skin aging.

FIG. 7 shows a histogram of the Integrated Optical Density (IOD)/Area values of Collagen XVII. As shown, the Blank (BC) shows the normal content of Collagen XVII without any treatment, as a benchmark for the experiment. The Negative Control (NC) showed a significant decrease in the content of Collagen XVII under UV irradiation, indicating that the experimental UV irradiation conditions resulted in a decrease in Collagen content, mimicking the skin damage or aging state. The positive control group (PC) had significantly increased content of Collagen XVII by known anti-aging treatment. Compared with a negative control group, the sample group (type 21 Collagen ColpepA1, 0.0125%) has significantly increased content of the Collagen XVII, and the lifting rate is 50.94%, which shows that ColpepA 121 effectively increases the content of the Collagen XVII.

The results show that the type 21 Collagen can effectively improve the content of the Collagen XVII in the skin, enhance the self-repairing capability of the skin and resist the influence of external environment factors, thereby resisting aging. This provides scientific support for type 21 collagen as an effective facial anti-aging filling ingredient.

Taken together, these positive effects exhibited by ColpepA a1 not only emphasize their potential value in promoting skin repair, enhancing skin moisturization, improving skin structural integrity, and anti-photoaging, but also reveals their great market potential as active ingredients for facial filling and anti-aging collagen materials. These experimental results provide a solid scientific basis for the use of ColpepA a1 in collagen materials for facial filling, demonstrating its important role in improving skin appearance, promoting skin health, and delaying aging.

Therefore, colpepA a 21 not only can be used as a high-efficiency facial filling component to promote the natural repair and regeneration of skin, but also can play a key role in the development of future anti-aging products, thereby providing innovative and effective solutions for consumers pursuing healthy and younger skin.

Example 4: preparation of composite injection of collagen ColpepA and 21

Materials:

type 21 collagen: 2% (w/v)

Physiological saline: in a sufficient amount as a solvent

Hyaluronic acid: 1% (w/v)

Vitamin C:0.05% (w/v) as antioxidant

The preparation method comprises the following steps:

Step 1: dissolving antioxidants

Vitamin C was first dissolved in a small amount of physiological saline to ensure complete dissolution.

Step 2: preparing collagen solution

The recombinant collagen powder was slowly added to the remaining physiological saline in a sterile container according to the desired final concentration. The mixture was stirred slowly at room temperature using a magnetic stirrer until the collagen was completely dissolved.

Step 3: adding hyaluronic acid

The pre-weighed hyaluronic acid was added to the collagen solution and stirring was continued to ensure uniform mixing until the hyaluronic acid was completely dissolved and a uniform viscous solution was formed.

Step 4: mixed antioxidant solution

Slowly adding the dissolved vitamin C solution into the solution containing collagen and hyaluronic acid, and continuously stirring until the whole solution is uniformly mixed.

Step 5: pH and viscosity adjustment

The pH of the solution is checked, if necessary adjusted to a pH range suitable for injection (about 7.0-7.4), the viscosity of the solution is adjusted, ensuring that it is suitable for subcutaneous injection, and has good flowability rather than being too dense.

Step 6: sterile filtration and filling

The solution was sterile filtered through a 0.22 micron sterile filter and the filtered solution was filled into pre-sterilized syringes under sterile conditions.

Comparative example 1: preparation of III type collagen composite injection

Materials:

Type III collagen: 2% (w/v)

Physiological saline: in a sufficient amount as a solvent

Hyaluronic acid: 1% (w/v)

Vitamin C:0.05% (w/v) as antioxidant

The preparation method is the same as in example 4.

Example 5: function of 21-type collagen composite injection on synthesis of human collagen

This example is directed to assessing the ability of type 21 collagen composite injections to promote Hydroxyproline (HYP) in human skin fibroblasts (HDF). HYP is an important component of collagen and can reflect the content of total collagen, so that the application potential of the injection in the face filling field can be estimated by measuring the content of HYP.

Experimental grouping:

Normal group (Control): without any treatment, this represents the level of collagen expression in the normal state of the cell.

H ₂O₂ modeling module (NC): cells were treated with H ₂O₂ to simulate oxidative damage due to oxidative stress.

Sample group a: on the basis of the modeling module, damaged cells were treated with 0.5% ColpepA 1.

Sample group B (positive control group): on the basis of the modeling module, damaged cells were treated with an equivalent amount of 0.5% type III collagen composite injection prepared using the method of comparative example 1.

Sample group C: on the basis of the modeling module, damaged cells were treated with an equivalent amount of 0.5% ColpepA% of the type 21 collagen composite injection prepared by the method of example 4.

HDF cell anti-aging test:

Cell inoculation: the cells are diluted to an inoculation density of 1X 10 ⁵/ml, inoculated into a 12-well plate according to a cell dilution of 1 ml/well, and incubated for 24+/-2 h in a cell incubator (37 ℃, 5% CO ₂, 95% RH); according to the experimental group, 3 duplicate wells were set per group.

Preparing liquid:

sample: dissolving the sample with a cell culture solution (the insoluble sample is added with DMSO to assist dissolution, but the final concentration of DMSO is not more than 0.5 percent), and diluting with the cell culture solution;

H ₂O₂ working solution: h ₂O₂ mother liquor is prepared into H ₂O₂ working solution by using a serum-free culture medium.

H ₂O₂ induction: and (3) sucking the cell culture medium in each group when the cell fusion degree in the 12-hole plate reaches 50% -70%, cleaning with PBS for one time, discarding and adding H ₂O₂ working solution.

Sample adding: after induction, PBS is washed for 2 times, and 1ml of culture solution is added into each hole of a normal group; 1ml of culture medium containing samples of the corresponding concentration was added to each well of the sample group. After the completion of the sample addition, the 12-well plate was placed in an incubator (37 ℃, 5% CO ₂, 95% RH) and cultured for 24.+ -. 2: 2 h.

And (3) detecting the HYP content: after cell culture 24.+ -.2 h, cells were collected and OD was read at 560nm following the HYP detection kit instructions.

Fig. 8 shows the HYP content data in the normal (Control), H ₂O₂ modeling (NC) and three sample groups. The results showed that the HYP level of the normal group (Control) was highest (22.71 ug/mL) as a baseline reference under normal physiological conditions. The HYP level of H ₂O₂ building blocks was significantly reduced to 6.98 ug/mL, indicating that H ₂O₂ successfully mimics the reduction in collagen synthesis due to oxidative stress. The HYP level (10.22 ug/mL) of sample group B (positive control group) using the type III collagen composite injection, although improved over the model group, showed some recovery of collagen, was still significantly lower than the control group. The HYP content of the sample group A added with 0.5% ColpepA1 is further improved than that of the sample group B, and the sample group A added with 0.5% 3835.1 is 12.01 ug/mL; the HYP level of the sample group C added with the type 21 collagen composite injection prepared by the method of the example 4 reaches 18.57 ug/mL, is obviously higher than that of other treatment groups and approaches to that of a control group, and shows the optimal collagen recovery capacity.

ColpepA 1A 21 of the compound injection (sample group C) showed the best collagen promoting effect, and particularly, it was remarkable in the recovery after the simulated oxidative damage. The special formula not only contains collagen, but also contains other components for promoting collagen synthesis or stabilization, such as hyaluronic acid and vitamin C, and is helpful for repairing and protecting skin. The use of the type 21 collagen composite injection, particularly in facial filling and anti-aging treatment, shows the capability of enhancing the collagen synthesis of skin, and has important value for improving the recovery of damaged skin and maintaining the elasticity and structural stability of the skin. These findings provide powerful scientific support for their clinical application and experimental basis for further development and optimization of anti-aging treatment strategies.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above description of the specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (13)

1. A collagen material for facial filling, wherein the collagen material comprises recombinant humanized collagen type 21, the amino acid sequence of which is selected from SEQ ID No.1, and further comprises an adjunct ingredient selected from one or more of: filling auxiliary agent, solvent, antioxidant, emulsifier, humectant, stabilizer, pH regulator and thickener.

2. The collagen material according to claim 1, wherein the concentration of collagen is 0.0001% -50% by total weight of the collagen material.

3. The collagen material according to claim 1, wherein the weight ratio of collagen to auxiliary components is selected from the group consisting of:

collagen and antibacterial agent: 10:1 to 30:1; or (b)

Collagen and anti-inflammatory agent: 15:1-100:1; or (b)

Collagen and growth factors: 100:1-300:1; or (b)

Collagen and humectant: 1:1 to 8:1; or (b)

Collagen and antioxidants: 10:1 to 50:1; or (b)

Collagen and emulsifying or thickening agents: 5:1 to 20:1 or a combination of the above.

4. A medical composition, wherein the composition comprises the collagen material of any one of claims 1-3.

5. The composition of claim 4, wherein the formulation of the composition comprises one or more of an injectable formulation, a topical application formulation, and a solution formulation.

6. The composition of claim 5, wherein the injectable formulation is selected from one or more of injectable hydrogels, composite injectables.

7. The composition of claim 6, wherein the injectable formulation is a collagen complex injection.

8. The composition of claim 5, wherein the topical application-type formulation is selected from one or more of an application gel, cream, or emulsion.

9. The composition of claim 5, wherein the solution formulation is selected from a collagen solution for a microneedle or microneedle roller.

10. Use of a recombinant humanized collagen material of type 21 for the preparation of a medical composition for facial filling, wherein the amino acid sequence of the collagen material comprises the amino acid sequence shown in SEQ ID No. 1.

11. The use of claim 10, wherein the medical composition comprises collagen material further comprising an adjunct ingredient selected from one or more of the following: filling auxiliary agent, solvent, antioxidant, emulsifier, humectant, stabilizer, pH regulator and thickener.

12. The use according to claim 11, wherein the collagen concentration is 0.0001% -50% by total weight of the collagen material comprised in the medical composition.

13. The use according to claim 11, wherein the medical composition comprises a collagen material comprising a collagen and an auxiliary component in a weight ratio selected from the group consisting of:

collagen and antibacterial agent: 10:1 to 30:1; or (b)

Collagen and anti-inflammatory agent: 15:1-100:1; or (b)

Collagen and growth factors: 100:1-300:1; or (b)

Collagen and humectant: 1:1 to 8:1; or (b)

Collagen and antioxidants: 10:1 to 50:1; or (b)

Collagen and emulsifying or thickening agents: 5:1 to 20:1 or a combination of the above.