Triple-helix structure collagen fleece membrane, preparation method therefor, and use thereof

Abstract

The present invention relates to the technical field of medical and cosmetic raw materials, and particularly relates to a triple-helix structure collagen fleece membrane, a preparation method therefor, and use thereof. In the present invention, without involving a chemical crosslinking agent, vacuum freeze-drying treatment is adopted, and the prepared triple-helix structure collagen fleece membrane possesses excellent mechanical properties and high production efficiency. Excellent biocompatibility ensures that the triple-helix structure collagen fleece membrane, when used in the preparation of medicines or cosmetics, does not cause immune reactions such as skin inflammation.

Description

Triple-helix collagen membrane, its preparation method and application

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202411715910.8, filed on November 27, 2024, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of medical and cosmetic raw material technology, specifically to a triple helix structure collagen membrane, its preparation method, and its application. Background Technology

[0004] Collagen is a major component of the extracellular matrix and is the most abundant and widely distributed protein in animals. Type I collagen is an important member of the human collagen family, accounting for over 90% of total collagen. It is mainly distributed in human tissues such as skin, bones, muscles, and blood vessels, playing a crucial role in maintaining the structure and function of these tissues. As a medical biomaterial, the differences between different species of Type I collagen are limited to minor variations in amino acid sequences or α-chain types and combinations; its basic structure and function are consistent with human Type I collagen. Therefore, its immunogenicity is relatively low, and it has high homology, making it easier for human tissues or cells to absorb and fill the spaces between the skin's matrix layers, resulting in fuller skin, smoother wrinkles, and increased skin density and tension. It can also repair damaged and sensitive skin tissue and promote wound healing and regeneration.

[0005] Collagen sponge manufacturing methods often employ freeze-drying and cross-linking processes. For example, CN101695581A discloses a freeze-dried sponge prepared using collagen and chitosan, cross-linked with glutaraldehyde solution. The patent describes that sponges obtained using only collagen have poor mechanical properties. CN112426560A discloses a method of placing semi-finished collagen sponge in a vacuum environment at 105℃-160℃ for 12-48 hours for vacuum heat treatment. After heat treatment, the water absorption rate, tensile strength, and porosity increase, while maintaining bioactivity. In actual production, the freeze-drying combined with thermal cross-linking treatment takes 60-96 hours, resulting in low production efficiency. CN106975099A discloses a method of chemically cross-linking and modifying collagen sponge raw materials during dialysis to form collagen gel. The resulting gel is then homogenized, injected into a suitable mold, and freeze-dried. This process is complex, and the raw material concentration is difficult to control consistently.

[0006] It can be seen that most collagen films in the existing technology are prepared by cross-linking with chemical cross-linking agents. Collagen films not prepared by chemical cross-linking agents often have problems such as poor mechanical properties and low production efficiency. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of poor mechanical properties and low production efficiency of collagen films prepared without the use of chemical cross-linking agents in the prior art, and to provide a triple helix collagen film that does not involve chemical cross-linking agents and has better mechanical properties and high production efficiency.

[0008] To achieve the above objectives, the present invention provides a triple-helix collagen velvet membrane, which does not contain a chemical crosslinking agent; and, when the thickness of the velvet membrane is 500-1000 μm, the stress range of the velvet membrane is 0.5-1.5 MPa and the strain range is 15-60%.

[0009] A second aspect of the present invention provides a method for preparing a triple-helix collagen velvet membrane, the method comprising:

[0010] Triple-helix collagen extracted from animal-derived collagen was dispersed in a solvent and subjected to a first vacuum freeze-drying process without the use of a chemical cross-linking agent to prepare the triple-helix collagen membrane.

[0011] The first vacuum freeze-drying process includes a first vacuum freeze-drying process and a first analytical drying process performed sequentially. The first analytical drying process is a three-stage process, wherein the first stage conditions include: a temperature of 100-130℃ and a time of 5-12 hours; the second stage conditions include: a temperature of 90-120℃ and a time of 5-12 hours; and the third stage conditions include: a temperature of 80-110℃ and a time of 5-12 hours.

[0012] The temperature of the first stage of processing > the temperature of the second stage of processing > the temperature of the third stage of processing.

[0013] The third aspect of the present invention provides a triple-helix collagen membrane prepared by the method for preparing a triple-helix collagen membrane according to the second aspect of the present invention.

[0014] The fourth aspect of this invention provides the application of the triple-helix collagen membrane described in the first or third aspect of this invention in the preparation of pharmaceuticals or cosmetics.

[0015] Beneficial effects of the invention

[0016] Without involving chemical cross-linking agents, the present invention uses the above-mentioned first vacuum freeze-drying process to prepare a triple-helix collagen membrane with excellent mechanical properties and high production efficiency. This allows it to be used in the preparation of pharmaceuticals or cosmetics, where it can both exert the loading effect of collagen on active ingredients and avoid causing immune reactions. Attached Figure Description

[0017] Figure 1 is the tensile force-tensile displacement change rate curve of the triple helix collagen velvet membrane prepared in Example 1;

[0018] Figure 2 shows the cytotoxicity test results of the control group and the triple-helix collagen velvet membrane prepared in Example 1 in Test Example 2. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described in detail below with reference to examples. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated components and steps, without excluding the presence of other substances or steps.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments.

[0022] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some embodiments, materials, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0023] Embodiments of the invention are described in detail below. However, these embodiments are exemplary, and the invention is not limited thereto; rather, the invention is defined by the scope of the claims.

[0024] The present invention provides a triple helix collagen velvet membrane, which does not contain a chemical crosslinking agent; and, when the thickness of the velvet membrane is 500-1000 μm, the stress range of the velvet membrane is 0.5-1.5 MPa and the strain range is 15-60%.

[0025] In this invention, the triple-helix collagen membrane does not involve chemical cross-linking agents and possesses superior mechanical properties. Even at a relatively thin thickness, it can maintain the aforementioned stress and strain characteristics. Particularly preferred is that, when the membrane thickness is 600-800 μm, the membrane stress ranges from 0.8 to 1.3 MPa, and the strain ranges from 20-45%. More preferably, the membrane stress ranges from 1.0 to 1.3 MPa, and the strain ranges from 35-45%. Therefore, the triple-helix collagen membrane of this invention, when used in pharmaceuticals or cosmetics, can effectively load active ingredients with collagen without causing an immune response.

[0026] In this invention, to further alleviate the immune response caused by the triple-helix collagen membrane to the human body, a triple-helix collagen membrane with high purity can be obtained. Preferably, the amino acid content of the membrane is not less than 90 wt%, and more preferably not less than 92 wt%. For example, the amino acid content of the membrane can be 92-98 wt%.

[0027] In this invention, the molecular weight of the triple-helix collagen in the velvet membrane can be appropriately selected within a wide range. Preferably, the molecular weight of the triple-helix collagen in the velvet membrane is 200-400 kDa, and more preferably 300-350 kDa. The triple-helix collagen forming the velvet membrane can be derived from various animal-derived collagens. Preferably, the triple-helix collagen is derived from one or more of bovine split skin, porcine split skin, horse split skin, bovine Achilles tendon, bovine hoof tendon, and beaver rat tail tendon, as detailed in the preparation method below.

[0028] In this invention, the triple helix collagen membrane can be prepared without the use of chemical cross-linking agents. To further reduce the immune response of the skin, preferably, the membrane does not contain emulsifiers and preservatives.

[0029] The second aspect of the present invention provides a method for preparing a triple-helix collagen membrane, the method comprising: dispersing triple-helix collagen extracted from animal-derived collagen in a solvent, and performing a first vacuum freeze-drying treatment without the use of a chemical cross-linking agent to prepare the triple-helix collagen membrane;

[0030] The first vacuum freeze-drying process includes a first vacuum freeze-drying process and a first analytical drying process performed sequentially. The first analytical drying process is a three-stage process, wherein the first stage conditions include: a temperature of 100-130℃ and a time of 5-12 hours; the second stage conditions include: a temperature of 90-120℃ and a time of 5-12 hours; and the third stage conditions include: a temperature of 80-110℃ and a time of 5-12 hours.

[0031] The temperature of the first stage of processing > the temperature of the second stage of processing > the temperature of the third stage of processing.

[0032] In this invention, preferably, the first stage of processing conditions includes: a temperature of 110-120℃ (e.g., 112℃, 115℃, 118℃) and a time of 6-10h; the second stage of processing conditions includes: a temperature of 100-110℃ (e.g., 102℃, 105℃, 108℃) and a time of 6-10h; the third stage of processing conditions includes: a temperature of 90-100℃ (e.g., 92℃, 95℃, 98℃) and a time of 6-10h; and the temperature of the first stage of processing is greater than the temperature of the second stage of processing, which is greater than the temperature of the third stage of processing.

[0033] In this invention, preferably, the conditions for the first vacuum freezing treatment include: a pressure of 1-3 mbar; a temperature of -20 to -70°C; and a time of 8-12 h; more preferably, the conditions for the first vacuum freezing treatment include: a pressure of 1-3 mbar; a temperature of -35 to -55°C; and a time of 6-10 h.

[0034] In this invention, the triple-helix collagen velvet membrane prepared by the above method without the use of chemical cross-linking agents still has excellent mechanical properties and will not cause skin immune response when applied to medicines or cosmetics. In order to further reduce skin immune response, according to a preferred embodiment, the preparation method of the triple-helix collagen velvet membrane does not use emulsifiers and preservatives.

[0035] In this invention, the method for extracting triple-helix collagen from animal-derived collagen can be prepared using the following method, which is more conducive to maintaining the triple-helix structure and purity of the obtained animal-derived protein. That is, in a preferred case, the preparation method of the above-mentioned triple-helix collagen membrane includes the following steps:

[0036] A1: Animal-derived collagen is pretreated to remove residual fat and polysaccharides;

[0037] A2: The pretreated product is then subjected to alkali treatment and acid treatment in sequence;

[0038] A3: The product after acid treatment is subjected to a second vacuum freeze-drying process to extract the triple helix collagen.

[0039] According to the present invention, in step A1, preferably, the pretreatment includes soaking the animal-derived collagen in a disinfectant solution for 10-60 minutes, preferably 10-30 minutes; more preferably, the temperature for soaking the raw material in the disinfectant solution is 20-40°C, preferably 20-30°C.

[0040] In some preferred embodiments, the pretreated raw materials soaked in disinfectant are placed in a salt solution and soaked for 15-30 hours, preferably 20-25 hours; more preferably, the temperature for soaking the raw materials in the salt solution is 1-10°C, preferably 3-8°C; in this invention, it should be understood that the above soaking usually requires the amount of soaking solution to basically cover the animal-derived collagen.

[0041] According to some preferred embodiments, to efficiently remove residual fats and polysaccharides, the disinfectant is selected from at least one of benzalkonium chloride, chlorhexidine acetate, and chlorhexidine, which are generally available by purchase; and,

[0042] The salt solution is preferably selected from at least one of NH4Cl-physiological saline solution, EDTA-physiological saline solution, and EDTA-phosphate buffer solution; more preferably, the concentration of the salt solution can be 0.2-0.5 mol / L, where the concentration mainly refers to the concentration of NH4Cl and EDTA in the solution. For example, 0.2-0.5 mol / L NH4Cl-physiological saline solution means that the concentration of NH4Cl in the NH4Cl-physiological saline solution is 0.2-0.5 mol / L; 0.2-0.5 mol / L EDTA-physiological saline solution means that the concentration of EDTA in the EDTA-physiological saline solution is 0.2-0.5 mol / L; 0.2-0.5 mol / L EDTA-phosphate buffer solution means that the concentration of EDTA in the EDTA-phosphate buffer solution is 0.2-0.5 mol / L.

[0043] In this invention, the animal-derived collagen can be appropriately selected from a variety of animal-derived collagens. Preferably, the animal-derived collagen is selected from one or more of bovine split skin, pig split skin, horse split skin, bovine Achilles tendon, bovine tendon, and beaver tail tendon.

[0044] According to the present invention, in step A2, in order to improve the efficiency of the alkali treatment and acid treatment, preferably, the alkali treatment includes: placing the pretreated product in an alkali solution, soaking it for 2-5 weeks, and then performing a washing treatment; the acid treatment includes: placing the alkali-treated product in an acid solution, soaking it for 10-20 hours, and then performing a washing treatment.

[0045] In this invention, preferably, the alkaline solution is selected from sodium hydroxide solution and / or potassium hydroxide solution; more preferably, the molar concentration of the alkaline solution is 0.1-0.9 mol / L, preferably 0.25-0.75 mol / L, for example, it can be 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, 0.55 mol / L, 0.6 mol / L, 0.65 mol / L, 0.7 mol / L, etc., and any range between these values.

[0046] In this invention, preferably, the acid solution is selected from hydrochloric acid solution and / or acetic acid solution; more preferably, the molar concentration of the acid solution is 0.3-1.2 mol / L, preferably 0.5-1.0 mol / L, for example, it can be 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, etc., and any range between these values. Similarly, it should be understood that the above soaking usually requires the amount of soaking solution to basically cover the animal-derived collagen to be treated.

[0047] According to the present invention, in step A2, preferably, in the alkali treatment, the washing process includes washing the raw material soaked in alkali solution with deionized water until the pH value of the deionized water reaches 6.5-7.5, for example, values ​​such as 6.5, 6.8, 7, 7.1, 7.5, and any range thereof; more preferably, in the acid treatment, the washing process includes washing the raw material soaked in acid solution with deionized water until the pH value of the deionized water reaches 2-4, for example, values ​​such as 2, 3, 3.2, 4, and any range thereof.

[0048] According to the present invention, in step A3, preferably, the second vacuum freeze-drying process includes a second vacuum freeze-drying process and a second desorption drying process.

[0049] In this invention, preferably, the conditions for the second vacuum freezing treatment include: a pressure of 1-3 mbar; a temperature of -20 to -70°C; and a time of 8-12 h; the conditions for the second analytical drying treatment include: a temperature of 90-120°C; and a time of 18-30 h. It can be understood that the second analytical drying treatment mainly employs a one-stage drying process.

[0050] According to the present invention, in step A3, preferably, the product A2 is subjected to grinding treatment before the vacuum freeze-drying treatment; more preferably, the grinding treatment conditions include: a speed of 1000-3000 rpm and a time of 10-30 min.

[0051] According to the present invention, in a preferred embodiment, the preparation method of dispersing the triple-helix collagen extracted from animal-derived collagen in a solvent comprises: dispersing the triple-helix collagen in an aqueous solvent (e.g., pure water, especially deionized water) to prepare a collagen dispersion solution of 10-50 mg / ml; more preferably, the mass concentration of the collagen dispersion solution is 15-20 mg / ml, for example, it can be 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, etc., and any range between these values.

[0052] According to the present invention, the first vacuum freeze-drying process in the preparation method may further include being carried out in the presence of a freeze-drying protectant, which may be selected from a variety of freeze-drying protectants. Preferably, the freeze-drying protectant is selected from one or more of mannitol, glycine, trehalose, polyethylene glycol, polyvinylpyrrolidone, thiols, liposomes, and protein polymers. More preferably, based on the amount of the triple-helix collagen protein, the amount of freeze-drying protectant added is 3wt%-25wt%, preferably 5wt%-12wt%, for example, it may be 5wt%, 8wt%, 10wt%, 12wt%, etc., and any range between these values.

[0053] According to the present invention, in a preferred embodiment, the first vacuum freeze-drying process in the preparation method further includes a sterilization process; more preferably, the sterilization process is selected from at least one of irradiation sterilization, ultraviolet sterilization and ethylene oxide sterilization.

[0054] The third aspect of the present invention provides a triple-helix collagen membrane prepared by the method for preparing a triple-helix collagen membrane according to the second aspect of the present invention.

[0055] The fourth aspect of this invention provides the application of the triple-helix collagen membrane described in the first or third aspect of this invention in the preparation of pharmaceuticals or cosmetics.

[0056] In this invention, the triple-helix collagen membrane has applications in the pharmaceutical field including hemostatic sponges, wound dressings, and artificial skin; and in cosmetics including facial masks, exosome-loaded transdermal applications, and post-cosmetic surgery repair treatments. The triple-helix collagen membrane of this invention exhibits high cell and tissue compatibility, is non-irritating and non-immunogenic in both pharmaceutical and cosmetic fields, and has a naturally good loading effect on active ingredients such as cell growth factors, exosomes, and liposomes, promoting their sustained release and achieving excellent repair effects. Simultaneously, the triple-helix collagen membrane of this invention possesses excellent mechanical properties.

[0057] The present invention will now be described in detail through examples, but the present invention is not limited to the examples described below. In the following examples, unless otherwise specified, all raw materials used are commercially available products.

[0058] Example 1

[0059] A1: At room temperature (25℃, the same below), 600g of bovine second layer hide was soaked in benzalkonium chloride solution for 10 minutes, and then placed in 0.2mol / L NH4Cl- physiological saline solution and soaked at 4℃ for 24h to complete the pretreatment of raw materials;

[0060] A2: At room temperature, the product after step A1 was soaked in 0.75 mol / L NaOH solution for 4 weeks and then washed with deionized water until the pH of the washing water was 6.8; the product after alkali treatment was soaked in 1.0 mol / L hydrochloric acid solution for 12 hours and then washed with deionized water until the pH of the washing water was 3.0.

[0061] A3: The product after step A2 is ground at 1500 rpm for 15 min and then subjected to a second vacuum freeze treatment and a second analytical drying treatment in sequence; wherein, the conditions for the second vacuum freeze treatment are: pressure of 1.5 mbar, temperature of -25℃, and time of 10 h; the conditions for the second analytical drying treatment are: temperature of 100℃ and time of 20 h.

[0062] A4: The product from step A3 was dispersed in deionized water to prepare a collagen dispersion solution with a mass fraction of 20 mg / mL. Then, 8 wt% (based on the mass of the triple-helix collagen protein) of mannitol was added and stirred for 4 hours. The dispersion solution was then allowed to stand at 4°C for 12 hours, followed by a first vacuum freeze treatment and a first desorption drying treatment. The conditions for the first vacuum freeze treatment were: pressure of 2 mbar, temperature of -50°C, and time of 6 hours. The first desorption drying treatment was carried out in three stages: the first stage was at 120°C for 6 hours; the second stage was at 110°C for 6 hours; and the third stage was at 100°C for 6 hours.

[0063] After the product undergoing the above-mentioned analysis and drying process was sterilized by irradiation, the triple helix collagen velvet membrane IA1 was prepared.

[0064] Example 2

[0065] A1: At room temperature (25℃, the same below), 500g of bovine second layer hide was soaked in benzalkonium chloride solution for 15 minutes, and then placed in 0.5mol / L NH4Cl- physiological saline solution and soaked at 5℃ for 22h to complete the pretreatment of raw materials;

[0066] A2: At room temperature, the product treated with A1 was soaked in 0.7 mol / L NaOH solution for 3 weeks and then washed with deionized water until the pH of the washing water was 7.1; the product treated with alkali was soaked in 0.8 mol / L hydrochloric acid solution for 10 hours and then washed with deionized water until the pH of the washing water was 3.2.

[0067] A3: The product after step A2 is ground at 2000 rpm for 15 min and then subjected to vacuum freeze-drying. The conditions for the second vacuum freeze-drying process are: pressure of 1.2 mbar, temperature of -22℃, and time of 12 h. The conditions for the second desorption and drying process are: temperature of 110℃ and time of 18 h.

[0068] A4: Disperse the triple helix collagen described in step A3 in deionized water to prepare a collagen dispersion solution with a mass fraction of 18 mg / mL. Then add 9 wt% (based on the mass of the triple helix collagen) of mannitol and stir for 4.5 h. After that, let the dispersion solution stand at 5°C for 10 h and then perform the first vacuum freeze treatment and the first desorption and drying treatment in sequence.

[0069] The conditions for the first vacuum freezing treatment include: a pressure of 3 mbar, a temperature of -45°C, and a time of 7 h; the first analytical drying treatment adopts a three-stage process, with the first stage treatment conditions including: a temperature of 120°C and a time of 7 h; the second stage treatment conditions including: a temperature of 110°C and a time of 7 h; and the third stage treatment conditions including: a temperature of 100°C and a time of 7 h.

[0070] After the product undergoing the above-mentioned analysis and drying process was sterilized by irradiation, the triple helix collagen velvet membrane IA2 was prepared.

[0071] Example 3

[0072] Following a similar method to Example 1, the difference lies in that the first analytical drying process in step A4 of this example is a three-stage process. The first stage processing conditions include: temperature of 100°C and time of 12h; the second stage processing conditions include: temperature of 90°C and time of 12h; the third stage processing conditions include: temperature of 80°C and time of 12h; the remaining steps are the same as in Example 1, and the triple helix collagen velvet membrane IA3 is prepared.

[0073] Example 4

[0074] Following a similar method to Example 1, the difference lies in that the first analytical drying process in step A4 of this example is a three-stage process. The first stage processing conditions include: temperature of 130°C and time of 5 hours; the second stage processing conditions include: temperature of 120°C and time of 5 hours; the third stage processing conditions include: temperature of 110°C and time of 5 hours; the remaining steps are the same as in Example 1, and the triple helix collagen velvet membrane IA4 is prepared.

[0075] Example 5

[0076] Following a similar method to Example 1, except that an equal weight of beaver rat tail tendon was used to replace bovine split skin to prepare the triple helix collagen velvet membrane IA5.

[0077] Example 6

[0078] The triple helix collagen velvet membrane IA6 was prepared by using a similar method as in Example 1, except that trehalose (purchased from Guangzhou Zhongguang Biotechnology Co., Ltd.) was used instead of mannitol in equal parts by weight.

[0079] Example 7

[0080] The triple-helix collagen membrane IA7 was prepared using a method similar to that in Example 1, except that a 0.1 mol / L NaOH solution was used instead of a 0.75 mol / L NaOH solution, and a 0.3 mol / L hydrochloric acid solution was used instead of a 1.0 mol / L hydrochloric acid solution.

[0081] Example 8

[0082] The triple-helix collagen velvet membrane IA8 was prepared by following a similar method to Example 1, except that a 0.9 mol / L NaOH solution was used instead of a 0.75 mol / L NaOH solution, and a 1.2 mol / L hydrochloric acid solution was used instead of a 1.0 mol / L hydrochloric acid solution.

[0083] Example 9

[0084] Following a similar method to Example 1, except that, based on the amount of the triple-helix collagen protein, 15 wt% mannitol was used to replace 8 wt% mannitol to prepare the triple-helix collagen membrane IA9.

[0085] Example 10

[0086] Following a similar method to Example 1, except that, based on the amount of the triple-helix collagen protein, 20 wt% mannitol was used to replace 8 wt% mannitol to prepare the triple-helix collagen membrane IA10.

[0087] Comparative Example 1

[0088] The method is the same as in Example 1, except that the first analytical drying process in step A4 of this comparative example is a one-stage process, with the following conditions: temperature of 120°C and time of 10h, to prepare the triple helix collagen velvet membrane IB1.

[0089] Comparative Example 2

[0090] The method is the same as in Example 1, except that the first analytical drying process in step A4 of this comparative example is a three-stage process. The first stage processing conditions include: temperature of 120°C and time of 2 hours; the second stage processing conditions include: temperature of 110°C and time of 2 hours; the third stage processing conditions include: temperature of 105°C and time of 2 hours; the remaining steps are the same as in Example 1, and the triple helix collagen velvet membrane IB2 is prepared.

[0091] Comparative Example 3

[0092] Referring to the method for preparing collagen sponges using glutaraldehyde solution as a chemical cross-linking agent disclosed in CN101695581A, the method includes:

[0093] A1: At room temperature (25℃, the same below), 600g of bovine head skin was soaked in benzalkonium chloride solution for 10 minutes, and then placed in NH4Cl-physiological saline solution (concentration of 0.5mol / L) and soaked at 4℃ for 24h to complete the pretreatment of raw materials;

[0094] A2: At room temperature, the product treated in step A1 was soaked in 0.75 mol / L NaOH solution for 4 weeks and then washed with deionized water until the pH of the washing water was 6.8; the product treated with alkali was soaked in 1.0 mol / L hydrochloric acid solution for 12 hours and then washed with deionized water until the pH of the washing water was 3.0.

[0095] A3: Dissolve the product of step A2 in distilled water to form a 1.0 wt% solution. Dissolve chitosan with a degree of deacetylation of 70% and a molecular weight of 200,000 Daltons in 1.0% acetic acid to form a 1.0 wt% solution. Then mix the two at a weight ratio of 5:1, add 2.0% glycerol of the total solution and stir until homogeneous. After vacuum freeze-drying, crosslink with 0.1% glutaraldehyde solution at room temperature for 10 hours. After crosslinking, wash several times with distilled water, and then vacuum freeze-dry and irradiate sterilize to prepare the triple helical collagen membrane IB3.

[0096] The vacuum freeze-drying conditions include vacuum freeze treatment and desorption drying treatment. The vacuum freeze treatment conditions include: a pressure of 2 mbar, a temperature of -50°C, and a time of 3 h. The desorption drying treatment conditions include: a temperature of 120°C and a time of 6 h.

[0097] Test Example 1

[0098] The amino acid content, thickness, stress range, and strain range of the triple helix collagen velvet membranes IA1-IA10 and IB1-IB3 prepared in Examples 1-10 and Comparative Examples 1-3 were tested respectively. The test results are shown in Table 1.

[0099] Test method:

[0100] 1. The total amino acid content of collagen chorionic membrane was determined by an amino acid analyzer.

[0101] 2. Place the collagen membrane into a vernier caliper, ensuring the sample does not easily fall out of the slot. Test and record 6 data points from the same membrane. Select 5 membranes prepared using the same process, measure and record the membrane thickness, for a total of 30 data points. Calculate all data, take the average, and calculate the error.

[0102] 3. Take 5-10 samples of each type of collagen membrane, cut them to a uniform length and width (4cm*1cm), and record the thickness data of each sample. Place the cut strips on a precision tensile testing instrument and record the initial displacement as S1. Start the instrument and stop it when the strip breaks. Record the displacement S2 and the maximum tensile force F at this point. Simultaneously, plot the tensile force and tensile displacement change rate test data of Example 1 into a curve, as shown in Figure 1. The tensile displacement change rate refers to the ratio of the change in strip displacement under tensile force to the initial displacement of the original strip. The tensile displacement change rate increases with the increase of tensile force. When the tensile force is the maximum tensile force F, the tensile force at this time is the measured stress, and the tensile displacement change rate is the measured strain.

[0103] Table 1

[0104] Test Example 2

[0105] The triple helix collagen chorionic membranes IA1-IA10 and IB1-IB3 prepared in Examples 1-10 and Comparative Examples 1-3 were tested for cytotoxicity and proliferation. Specifically, the indirect cytotoxicity of the chorionic membranes and their role as chorionic membranes as three-dimensional cell culture scaffold materials in promoting fibroblast proliferation were tested. The test results are shown in Table 2.

[0106] Test method:

[0107] 1. Cytotoxicity Assay (Indirect Toxicity): Collagen fibrous membranes were immersed in sterile cell culture medium for 24 hours at 37°C to prepare the extract. 100 μL of the extract was added to each well of a 96-well plate to culture L929 fibroblasts. Cells cultured in normal medium served as a control group. Cell viability was tested on days 1, 3, and 5 after culture. Simultaneously, the fibroblast survival in the cytotoxicity assay was recorded for both the triple-helix collagen fibrous membrane extract prepared in Example 1 and the control group, as shown in Figure 2.

[0108] 2. The effect of collagen chorionic villus membrane as a scaffold material for three-dimensional cell culture on promoting fibroblast proliferation: Using normally cultured cells in a well plate as a positive control group, collagen chorionic villus membrane was placed in the well plate as a three-dimensional cell culture scaffold. An equal number of cell suspensions were added to the culture plate and the collagen chorionic villus membrane. After 72 hours of culture, CCK-8 reagent was added at 24 hours and 72 hours after culture to test the absorbance value of the cell solution.

[0109] Table 2

[0110] As shown in Table 1, the triple-helix collagen velvet membranes prepared using Examples 1-10 of this invention possess excellent mechanical properties and an amino acid content of not less than 90%. Furthermore, in tests of indirect cytotoxicity and the effect of the velvet membranes as a scaffold material for three-dimensional cell culture in promoting fibroblast proliferation, Examples 1-10 exhibited similar effects to the control group. Therefore, the triple-helix collagen velvet membranes prepared by this invention possess excellent mechanical properties and, in pharmaceutical or cosmetic applications, can effectively load active ingredients with collagen without causing immune responses.

[0111] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A triple-helix collagen velvet membrane, characterized in that, The textured film does not contain chemical crosslinking agents; and, when the textured film thickness is 500-1000 μm, the textured film stress ranges from 0.5 to 1.5 MPa, and the strain ranges from 15% to 60%.

2. The fleece film according to claim 1, wherein, When the thickness of the velvet film is 600-800 μm, the stress range of the velvet film is 0.8-1.3 MPa, and the strain range is 20-45%.

3. The fleece film according to claim 1 or 2, wherein, The amino acid content of the velvet membrane is not less than 90 wt%, preferably not less than 92 wt%; And / or, the molecular weight of the triple-helix collagen in the velvet membrane is 200-400 kDa, preferably 300-350 kDa; And / or, the velvet film does not contain emulsifiers and preservatives.

4. A method for preparing a triple-helix collagen velvet membrane, characterized in that, The method includes: Triple-helix collagen extracted from animal-derived collagen was dispersed in a solvent and subjected to a first vacuum freeze-drying process without the use of a chemical cross-linking agent to prepare the triple-helix collagen membrane. The first vacuum freeze-drying process includes a first vacuum freeze-drying process and a first analytical drying process performed sequentially. The first analytical drying process is a three-stage process, wherein the first stage conditions include: a temperature of 100-130℃ and a time of 5-12 hours; the second stage conditions include: a temperature of 90-120℃ and a time of 5-12 hours; and the third stage conditions include: a temperature of 80-110℃ and a time of 5-12 hours. The temperature of the first stage of processing > the temperature of the second stage of processing > the temperature of the third stage of processing.

5. The preparation method according to claim 4, wherein, The first analytical drying process adopts a three-stage process. The first stage processing conditions include: temperature of 110-120℃ and time of 6-10h; the second stage processing conditions include: temperature of 100-110℃ and time of 6-10h; and the third stage processing conditions include: temperature of 90-100℃ and time of 6-10h.

6. The preparation method according to claim 4 or 5, wherein, The conditions for the first vacuum freezing treatment include: a pressure of 1-3 mbar; a temperature of -20 to -70°C; and a time of 8-12 h.

7. The preparation method according to any one of claims 4-6, wherein, This method does not use emulsifiers or preservatives.

8. The preparation method according to any one of claims 4-7, wherein, The extraction of triple-helix collagen from animal-derived collagen includes the following steps: A1: Animal-derived collagen is pretreated to remove residual fat and polysaccharides; A2: The pretreated product is then subjected to alkali treatment and acid treatment in sequence; A3: The product after acid treatment is subjected to a second vacuum freeze-drying process to extract the triple helix collagen.

9. The preparation method according to claim 8, wherein, In step A1, the pretreatment includes first soaking the animal-derived collagen in a disinfectant solution for 10-60 minutes, and then soaking it in a saline solution for 15-30 hours; Preferably, the disinfectant is selected from at least one of benzalkonium chloride solution, chlorhexidine acetate, and chlorhexidine; the salt solution is selected from at least one of NH4Cl-physiological saline solution, EDTA-physiological saline solution, and EDTA-phosphate buffer solution. Preferably, the animal-derived collagen is selected from one or more of the following: bovine split skin, porcine split skin, horse split skin, bovine Achilles tendon, bovine hoof tendon, and beaver rat tail tendon.

10. The preparation method according to claim 8 or 9, wherein, In step A2, the alkali treatment includes: placing the pretreated product in an alkali solution and soaking it for 2-5 weeks, followed by washing; the acid treatment includes: placing the alkali-treated product in an acid solution and soaking it for 10-20 hours, followed by washing. Preferably, the alkaline solution is selected from sodium hydroxide solution and / or potassium hydroxide solution; more preferably, the molar concentration of the alkaline solution is 0.1-0.9 mol / L, preferably 0.25-0.75 mol / L; Preferably, the acid solution is selected from hydrochloric acid solution and / or acetic acid solution; more preferably, the molar concentration of the acid solution is 0.3-1.2 mol / L, and more preferably 0.5-1.0 mol / L.

11. The preparation method according to any one of claims 8-10, wherein, In step A3, the second vacuum freeze-drying process includes a second vacuum freeze-drying process and a second desorption drying process; The conditions for the second vacuum freezing treatment include: a pressure of 1-3 mbar; a temperature of -20 to -70°C; and a time of 8-12 hours. The conditions for the second analytical drying process include: a temperature of 90-120℃ and a time of 18-30h.

12. The preparation method according to any one of claims 4-11, wherein, The vacuum freeze-drying process is carried out in the presence of a freeze-drying protectant. Preferably, the freeze-drying protectant is selected from one or more of mannitol, glycine, trehalose, polyethylene glycol, polyvinylpyrrolidone, thiols, liposomes, and protein polymers; Preferably, based on the amount of the triple-helix collagen protein, the amount of the freeze-drying protectant added is 3wt%-25wt%, more preferably 5wt%-12wt%.

13. The preparation method according to any one of claims 4-12, wherein, The first vacuum freeze-drying process also includes sterilization. Preferably, the sterilization treatment is selected from at least one of irradiation sterilization, ultraviolet sterilization, and ethylene oxide sterilization.

14. A triple-helix collagen velvet membrane prepared by the preparation method according to any one of claims 4-13.

15. The use of a triple-helix collagen membrane according to any one of claims 1-3 and 14 in the preparation of pharmaceuticals or cosmetics.

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