Collagen-containing composition and application thereof in preparation of injection filling material

Abstract

The invention belongs to the technical field of filling materials, and particularly discloses a collagen-containing composition and application thereof in preparation of an injection filling material, and the collagen-containing composition comprises the following components in percentage by weight: 0.1-0.3% of serine, 0.2-0.6% of ergothioneine, 1.2-2% of recombinant human collagen, 1.5-2% of swimming bladder collagen, 2-4% of modified hyaluronic acid, 0.4-1% of a cross-linking agent and the balance of a PBS (Phosphate Buffer Solution). The collagen (the recombinant human collagen and the swimming bladder collagen are synergistic) and the modified hyaluronic acid form a stable three-dimensional network structure under the action of the cross-linking agent, so that the material has relatively high storage modulus and relatively low loss modulus. Therefore, the composition disclosed by the invention has important application in the field of biomedicine, especially skin repair and tissue engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of filling materials, and particularly relates to a collagen-containing composition and its application in the preparation of injectable filling materials. Background Art

[0002] With the development of medical aesthetics and regenerative medicine technologies, injectable filling materials, as an important treatment method, have been widely used in the treatment of problems such as skin aging and soft tissue defects. However, existing injectable filling materials mostly have problems of relatively low storage modulus and high loss modulus, resulting in difficulty in maintaining long-term stability during use and possibly causing tissue rejection reactions, affecting the curative effect. At the same time, the cell proliferation effect of existing materials is relatively limited and cannot well support tissue repair and regeneration.

[0003] Collagen is an important component of the skin and connective tissues, with good biocompatibility and biodegradability, and has become an ideal choice for injectable filling materials. However, traditional collagen materials usually have deficiencies in physical properties and cell proliferation.

[0004] In view of this, the present application is proposed. Summary of the Invention

[0005] The present invention provides a collagen-containing composition and its application in the preparation of injectable filling materials. The composition of the present invention has excellent storage modulus and low loss modulus, and can effectively increase the cell proliferation rate and promote the regeneration of collagen.

[0006] The present invention solves its technical problems by adopting the following technical solutions: A collagen-containing composition, comprising the following components in percentage by weight: 0.1 - 0.3% serine, 0.2 - 0.6% ergothioneine, 1.2 - 2% recombinant human collagen, 1.5 - 2% fish swim bladder collagen, 2 - 4% modified hyaluronic acid, 0.4 - 1% cross-linking agent, and the balance PBS buffer.

[0007] Serine: Serine is a common amino acid in the human body and has various physiological functions. In the collagen composition, serine supports cell attachment and proliferation by promoting the interaction between cells and the matrix. It can enhance the binding force between collagen and cells, improve the cell growth environment, and thus increase the cell proliferation rate. In addition, the introduction of serine can regulate the three-dimensional structure of collagen and enhance its biocompatibility and biological activity.

[0008] Ergothioneine: Ergothioneine is a natural antioxidant that can scavenge free radicals in cells and reduce the damage of oxidative stress to cells. It has a significant impact on the anti-aging, anti-inflammatory and repair effects of cells, especially in promoting the proliferation and repair of skin cells, and can improve the stability of the formulation to a certain extent.

[0009] Recombinant human collagen: Recombinant human collagen not only provides structural support for cells, but also stimulates cell proliferation, migration and differentiation. By binding to cell surface receptors, it promotes the activation of cell signaling pathways, thereby enhancing cell proliferation and repair capabilities.

[0010] Fish bladder collagen: Fish bladder collagen is collagen extracted from the bladder tissue of fish and is rich in collagen. Its structure is similar to that of collagen in human skin and can provide structural support and repair for the skin. Fish bladder collagen forms a network structure to enhance the stability of the entire collagen matrix and promote cell adhesion and proliferation. In addition, it can also promote wound healing and repair and improve the skin barrier function.

[0011] Modified hyaluronic acid: Hyaluronic acid has good biocompatibility. In this formulation, modified hyaluronic acid helps to enhance the stability and physical properties of the composition. Modified hyaluronic acid can enhance the mechanical properties of the gel composition, increase the storage modulus, while reducing the loss modulus, and improve the elasticity and toughness of the material. In addition, through modification, modified hyaluronic acid participates in processes such as cell adhesion, migration, proliferation and differentiation. It is more suitable for cell growth, thereby improving the cell microenvironment and promoting cell proliferation.

[0012] Cross-linking agent: The addition of a cross-linking agent can enhance the cross-linking between collagen and other components to form a more stable and firm network structure. This cross-linked structure helps to increase the storage modulus (i.e., the elasticity of the material) of the composition and control its loss modulus (i.e., the viscosity of the material). In terms of cell growth, the cross-linked structure can provide a stable support structure to help cells grow on the surface of the material and promote cell proliferation.

[0013] Among them, collagen (synergistic effect of recombinant human collagen and fish bladder collagen) and modified hyaluronic acid form a stable three-dimensional network structure under the action of a cross-linking agent, making the material have a high storage modulus and a low loss modulus. Thus, the composition of the present invention has important applications in the biomedical field, especially in skin repair and tissue engineering.

[0014] Among them, components such as serine, ergothioneine, collagen (recombinant human collagen and fish swim bladder collagen), and modified hyaluronic acid act together to provide an ideal proliferation environment for cells. In particular, collagen stimulates cell proliferation and migration by binding to cell surface receptors; while serine and ergothioneine further increase the cell proliferation rate by enhancing the cell's antioxidant stress tolerance. Moreover, the components in this composition not only provide structural support but also promote wound repair and tissue regeneration by stimulating cell proliferation and differentiation. In particular, the combination of recombinant human collagen and fish swim bladder collagen enhances the biocompatibility of the material and improves the tissue repair effect.

[0015] As a preferred embodiment of the present invention, the crosslinking agent comprises polycarboxylic acid and D-ribose with a mass ratio of 1:(0.5 - 2); the polycarboxylic acid is malic acid.

[0016] As a preferred embodiment of the present invention, the preparation method of the fish swim bladder collagen is as follows: (1) Crush the fish swim bladder of grass carp to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous glutaraldehyde solution, add cellulase, β-glucanase, ficin, and sodium chloride, and carry out enzymatic hydrolysis to obtain an enzymatic hydrolysate; (3) First filter the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then filter it through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and dry it to obtain fish swim bladder collagen.

[0017] As a preferred embodiment of the present invention, the mass ratio of the fish swim bladder powder to the aqueous glutaraldehyde solution is 1:(4 - 12); The mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 0.5 - 1.2%.

[0018] As a preferred embodiment of the present invention, the mass ratio of the fish swim bladder powder, cellulase, β-glucanase, ficin, and sodium chloride is 1:(0.01 - 0.02):(0.005 - 0.012):(0.002 - 0.006):(0.001 - 0.002).

[0019] As a preferred embodiment of the present invention, the preparation method of the modified hyaluronic acid is as follows: (1) Add hyaluronic acid to an aqueous sodium dodecylbenzenesulfonate solution, stir evenly, add 1-ethyl-3-methylimidazolium acetate, and stir evenly to obtain a mixed solution; (2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjust the pH to 5.4 - 5.8, react at 55 - 70 °C for 2 - 6 h, and dry to obtain modified hyaluronic acid.

[0020] As a preferred embodiment of the present invention, the mass ratio of hyaluronic acid, sodium dodecylbenzenesulfonate aqueous solution, and 1-ethyl-3-methylimidazolium acetate is 1:(5 - 12):(0.05 - 0.12).

[0021] As a preferred embodiment of the present invention, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is (0.04 - 0.08):(0.04 - 0.08):1.

[0022] As a preferred embodiment of the present invention, the mass percentage content of sodium dodecylbenzenesulfonate in the sodium dodecylbenzenesulfonate aqueous solution is 0.5 - 1.2%.

[0023] The present invention also provides the application of a collagen-containing composition in the preparation of an injectable filling material.

[0024] Advantages of the present invention: The collagen of the present invention (synergistic effect of recombinant human-derived collagen and fish swim bladder collagen) and modified hyaluronic acid form a stable three-dimensional network structure under the action of a cross-linking agent, making the material have a relatively high storage modulus and a relatively low loss modulus. Therefore, the composition of the present invention has important applications in the biomedical field, especially in skin repair and tissue engineering. Specific Embodiments

[0025] 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. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] In the present invention, among the technically characterized descriptions in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0027] In the present invention, regarding the numerical range, unless otherwise specified, the above numerical range is considered continuous and includes the minimum value and the maximum value of this range, as well as each value between such minimum value and maximum value. Further, when the range refers to an integer, it includes each integer between the minimum value and the maximum value of this range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0028] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0029] Unless otherwise specified, all reagents or instruments used in the present invention are conventional products that can be obtained commercially. The raw materials used in each pair of ratios and the raw materials used in the parallel experiments of each example are the same commercially available products unless otherwise specified.

[0030] Example 1 A collagen-containing composition, comprising the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 1.8% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0031] The crosslinking agent comprises malic acid and D-ribose in a mass ratio of 1:1.

[0032] The recombinant human collagen is derived from Medhao, with the brand number MDH.

[0033] The preparation method of the fish swim bladder collagen is as follows: (1) Clean the fish swim bladder of grass carp, dry it, and pulverize it to 100 mesh to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous glutaraldehyde solution, add cellulase, β-glucanase, ficin, and sodium chloride, and enzymatically hydrolyze at 50 °C for 4.5 h to obtain an enzymatic hydrolysate; The mass ratio of the fish swim bladder powder, aqueous glutaraldehyde solution, cellulase, β-glucanase, ficin, and sodium chloride is 1:10:0.02:0.005:0.006:0.001.

[0034] The mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 1%.

[0035] (3) First filter the enzymatic hydrolysate with an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then filter it with an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and dry it to obtain fish swim bladder collagen.

[0036] The preparation method of the modified hyaluronic acid is as follows: (1) Add hyaluronic acid to an aqueous sodium dodecylbenzenesulfonate solution, stir evenly, add 1-ethyl-3-methylimidazolium acetate, and stir evenly to obtain a mixed solution; The mass ratio of the hyaluronic acid, aqueous sodium dodecylbenzenesulfonate solution, and 1-ethyl-3-methylimidazolium acetate is 1:5:0.05.

[0037] The mass percentage content of sodium dodecylbenzenesulfonate in the aqueous sodium dodecylbenzenesulfonate solution is 1%.

[0038] (2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjust the pH to 5.6, react at 60 °C for 4 h, and dry to obtain modified hyaluronic acid. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is 0.06:0.06:1.

[0039] The preparation method of the collagen-containing composition described above includes the following steps: Add serine, ergothioneine, recombinant human collagen, and fish swim bladder collagen to PBS buffer, stir evenly, homogenize at 800 rpm for 10 min, then add modified hyaluronic acid and a crosslinking agent, and stir evenly to obtain the collagen-containing composition.

[0040] Example 2 A collagen-containing composition, comprising the following components in percentage by weight: 0.3% serine, 0.2% ergothioneine, 2% recombinant human collagen, 1.5% fish swim bladder collagen, 4% modified hyaluronic acid, 0.4% crosslinking agent, and the balance PBS buffer.

[0041] The crosslinking agent comprises malic acid and D-ribose with a mass ratio of 1:1.

[0042] The recombinant human collagen is derived from Maidehao, with the brand number MDH.

[0043] The preparation method of the fish swim bladder collagen is as follows: (1) Clean the fish swim bladder of grass carp, dry it, and crush it to 100 meshes to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous glutaraldehyde solution, add cellulase, β-glucanase, ficin, and sodium chloride, and enzymatically hydrolyze at 50 °C for 4.5 h to obtain an enzymatic hydrolysate; The mass ratio of the fish swim bladder powder, aqueous glutaraldehyde solution, cellulase, β-glucanase, ficin, and sodium chloride is 1:10:0.02:0.005:0.006:0.001.

[0044] The mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 1%.

[0045] (3) First filter the enzymatic hydrolysate with an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then filter it with an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and dry to obtain fish swim bladder collagen.

[0046] The preparation method of the modified hyaluronic acid is as follows: (1) Add hyaluronic acid to an aqueous sodium dodecylbenzenesulfonate solution, stir evenly, add 1-ethyl-3-methylimidazolium acetate, and stir evenly to obtain a mixed solution; The mass ratio of the hyaluronic acid, sodium dodecylbenzenesulfonate aqueous solution, and 1-ethyl-3-methylimidazolium acetate is 1:5:0.05.

[0047] The mass percentage of sodium dodecylbenzenesulfonate in the sodium dodecylbenzenesulfonate aqueous solution is 1%.

[0048] (2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjust the pH to 5.6, react at 60°C for 4 h, and dry to obtain modified hyaluronic acid. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is 0.06:0.06:1.

[0049] The preparation method of the collagen-containing composition includes the following steps: Add serine, ergothioneine, recombinant human collagen, and fish swim bladder collagen to PBS buffer, stir evenly, homogenize at 800 rpm for 10 min, then add modified hyaluronic acid and a crosslinking agent, and stir evenly to obtain the collagen-containing composition.

[0050] Example 3 A collagen-containing composition includes the following components in weight percentage: 0.3% serine, 0.6% ergothioneine, 1.2% recombinant human collagen, 2% fish swim bladder collagen, 2% modified hyaluronic acid, 1% crosslinking agent, and the balance PBS buffer.

[0051] The crosslinking agent includes malic acid and D-ribose with a mass ratio of 1:1.

[0052] The recombinant human collagen is sourced from Maidhao, with the product number MDH.

[0053] The preparation method of the fish swim bladder collagen is as follows: (1) Clean the fish swim bladder of grass carp, dry it, and pulverize it to 100 mesh to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous glutaraldehyde solution, add cellulase, β-glucanase, ficin, and sodium chloride, and enzymatically hydrolyze at 50°C for 4.5 h to obtain an enzymatic hydrolysate; The mass ratio of the fish swim bladder powder, aqueous glutaraldehyde solution, cellulase, β-glucanase, ficin, and sodium chloride is 1:10:0.02:0.005:0.006:0.001.

[0054] The mass percentage of glutaraldehyde in the aqueous glutaraldehyde solution is 1%.

[0055] (3) Filter the enzymatic hydrolysate first through an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and dry to obtain fish swim bladder collagen.

[0056] The preparation method of the modified hyaluronic acid is as follows: (1) Add hyaluronic acid to an aqueous solution of sodium dodecylbenzenesulfonate, stir evenly, add 1-ethyl-3-methylimidazolium acetate, and stir evenly to obtain a mixed solution; The mass ratio of the hyaluronic acid, the aqueous solution of sodium dodecylbenzenesulfonate, and 1-ethyl-3-methylimidazolium acetate is 1:5:0.05.

[0057] The mass percentage content of sodium dodecylbenzenesulfonate in the aqueous solution of sodium dodecylbenzenesulfonate is 1%.

[0058] (2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjust the pH to 5.6, react at 60 °C for 4 h, and dry to obtain modified hyaluronic acid. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is 0.06:0.06:1.

[0059] The preparation method of the collagen-containing composition includes the following steps: Add serine, ergothioneine, recombinant human collagen, and fish swim bladder collagen to PBS buffer, stir evenly, homogenize at 800 rpm for 10 min, then add modified hyaluronic acid and a cross-linking agent, and stir evenly to obtain a collagen-containing composition.

[0060] Example 4 A collagen-containing composition, comprising the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 1.8% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% cross-linking agent, and the balance PBS buffer.

[0061] The cross-linking agent includes malic acid and D-ribose with a mass ratio of 1:1.

[0062] The recombinant human collagen is derived from Maidhao, with the brand number MDH.

[0063] The preparation method of the fish swim bladder collagen is as follows: (1) Clean the fish swim bladder of grass carp, dry it, and crush it to 100 mesh to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous solution of glutaraldehyde, add cellulase, β-glucanase, ficin, and sodium chloride, and enzymatically hydrolyze at 50 °C for 4.5 h to obtain an enzymatic hydrolysate; The mass ratio of the swim bladder powder, glutaraldehyde aqueous solution, cellulase, β-glucanase, ficin, and sodium chloride is 1:10:0.01:0.012:0.002:0.002.

[0064] The mass percentage of glutaraldehyde in the glutaraldehyde aqueous solution is 1%.

[0065] (3) First filter the enzymatic hydrolysate with an ultrafiltration membrane having a molecular weight cut-off of 500 Da, then filter it with an ultrafiltration membrane having a molecular weight cut-off of 5000 Da, and dry it to obtain fish bladder collagen.

[0066] The preparation method of the modified hyaluronic acid is as follows: (1) Add hyaluronic acid to the sodium dodecylbenzenesulfonate aqueous solution, stir evenly, add 1-ethyl-3-methylimidazolium acetate, and stir evenly to obtain a mixed solution; The mass ratio of the hyaluronic acid, sodium dodecylbenzenesulfonate aqueous solution, and 1-ethyl-3-methylimidazolium acetate is 1:5:0.05.

[0067] The mass percentage of sodium dodecylbenzenesulfonate in the sodium dodecylbenzenesulfonate aqueous solution is 1%.

[0068] (2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjust the pH to 5.6, react at 60 °C for 4 h, and dry to obtain the modified hyaluronic acid. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is 0.06:0.06:1.

[0069] The preparation method of the collagen-containing composition includes the following steps: Add serine, ergothioneine, recombinant human collagen, and fish bladder collagen to PBS buffer, stir evenly, homogenize at 800 rpm for 10 min, then add the modified hyaluronic acid and crosslinking agent, and stir evenly to obtain the collagen-containing composition.

[0070] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain recombinant human collagen, and the others are the same.

[0071] A collagen-containing composition, comprising the following components in weight percentage: 0.2% serine, 0.5% ergothioneine, 1.8% fish bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0072] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain fish bladder collagen, and the others are the same.

[0073] A collagen-containing composition, comprising the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0074] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain recombinant human collagen (the total amount of recombinant human collagen and fish swim bladder collagen remains unchanged), and the others are the same.

[0075] A collagen-containing composition, comprising the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 3.4% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0076] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain fish swim bladder collagen (the total amount of recombinant human collagen and fish swim bladder collagen remains unchanged), and the others are the same.

[0077] A collagen-containing composition, comprising the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 3.4% recombinant human collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0078] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the preparation method of fish swim bladder collagen in Comparative Example 5 is different from that in Example 1, and the others are the same.

[0079] The preparation method of the fish swim bladder collagen is as follows: (1) Clean, dry, and pulverize the fish swim bladder of grass carp to 100 mesh to obtain fish swim bladder powder; (2) Add the fish swim bladder powder to an aqueous glutaraldehyde solution, add cellulase and pectinase, and enzymatically hydrolyze at 50 °C for 4.5 h to obtain an enzymatic hydrolysate; The mass ratio of the fish swim bladder powder, aqueous glutaraldehyde solution, cellulase, and pectinase is 1:10:0.02:0.005.

[0080] The mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 1%.

[0081] (3) First filter the enzymatic hydrolysate with an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then filter it with an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and dry it to obtain fish swim bladder collagen.

[0082] Comparative Example 6

[0083] The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, an equal amount of hyaluronic acid is used to replace the modified hyaluronic acid, and the others are the same.

[0084] A collagen-containing composition comprises the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 1.8% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0085] Comparative Example 7

[0086] The difference between Comparative Example 7 and Comparative Example 1 is that the preparation method of the modified hyaluronic acid is different, and the others are the same.

[0087] The preparation method of the modified hyaluronic acid is as follows: (1) Dispersing hyaluronic acid in water to obtain a mixed solution; The mass ratio of the hyaluronic acid to water is 1:5:0.05.

[0088] (2) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the mixed solution, adjusting the pH to 5.6, reacting at 60°C for 4 h, and drying to obtain the modified hyaluronic acid. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is 0.06:0.06:1.

[0089] Comparative Example 8

[0090] The difference between Comparative Example 8 and Example 1 is that the crosslinking agent is a single malic acid, and the others are the same.

[0091] A collagen-containing composition comprises the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 1.8% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0092] The crosslinking agent is malic acid.

[0093] Comparative Example 9

[0094] The difference between Comparative Example 9 and Example 1 is that the crosslinking agent is a single D-ribose, and the others are the same.

[0095] A collagen-containing composition comprises the following components in percentage by weight: 0.2% serine, 0.5% ergothioneine, 1.6% recombinant human collagen, 1.8% fish swim bladder collagen, 3.2% modified hyaluronic acid, 0.6% crosslinking agent, and the balance PBS buffer.

[0096] The cross-linking agent is D-ribose.

[0097] Test Example 1. The storage modulus and loss modulus were measured at 25 °C using a dynamic mechanical analysis (DMA) device.

[0098] 2. Cell proliferation experiment.

[0099] Take L929 cells in the logarithmic growth phase, transfer the culture dish containing L929 cells to the laminar flow hood, add trypsin and digest for 80 s. After the cells detach, use DMEM medium (containing 10% serum) to terminate the digestion. Collect the cells and perform cell counting. Inoculate the cells into a 96-well plate, with 10 4 cells per well. Set 3 replicate experimental wells in each group and culture overnight in an incubator at 37 °C and 5% CO2. When the cells adhere well, aspirate the original medium, add the corresponding extract in each group (add the original medium in the blank control group). The experimental group is the material + cells, the control group is the medium + cells, and the blank control group is the medium. Continue to culture in an incubator at 37 °C and 5% CO2 for 24 hours.

[0100] Before detection, aspirate the original extract (medium), add 200 μL of medium mixed with 20 μL of MTT reagent to each well (containing 10% serum and 1% double antibody), continue to incubate in an incubator at 37 °C and 5% CO2 for 4 hours. Then aspirate the liquid in the wells again, add 150 μL of dimethyl sulfoxide, shake for 10 minutes. When the color of the solution in each well is uniform and stable, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance (OD) value at a wavelength of 490 nm. Calculate the cell proliferation rate using the following formula: (average OD of the experimental group / average OD of the blank control group) × 100%.

[0101] Table 1

[0102] As can be seen from Table 1, the composition described in the present invention has excellent storage modulus and low loss modulus, and can effectively improve the cell proliferation rate and promote the regeneration of collagen.

[0103] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A collagen-containing composition, comprising the following components in percentage by weight: 0.1-0.3% serine, 0.2-0.6% ergothioneine, 1.2-2% recombinant human collagen, 1.5-2% fish swim bladder collagen, 2-4% modified hyaluronic acid, 0.4-1% crosslinking agent, and the balance being PBS buffer solution.

2. The collagen-containing composition according to claim 1, wherein The crosslinking agent comprises a polycarboxylic acid and D-ribose with a mass ratio of 1: (0.5-2); the polycarboxylic acid is malic acid.

3. The collagen-containing composition according to claim 1, wherein The preparation method of the fish swim bladder collagen is as follows: (1) Crushing the fish swim bladder of grass carp to obtain fish swim bladder powder; (2) Adding the fish swim bladder powder into an aqueous glutaraldehyde solution, adding cellulase, β-glucanase, ficin and sodium chloride, and performing enzymatic hydrolysis to obtain an enzymatic hydrolysate; (3) Filtering the enzymatic hydrolysate first with an ultrafiltration membrane with a molecular weight cut-off of 500 Da, then filtering with an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and drying to obtain fish swim bladder collagen.

4. The collagen-containing composition according to claim 3, wherein The mass ratio of the fish swim bladder powder to the aqueous glutaraldehyde solution is 1: (4-12); The mass percentage content of glutaraldehyde in the aqueous glutaraldehyde solution is 0.5-1.2%.

5. The collagen-containing composition according to claim 3, wherein The mass ratio of the fish swim bladder powder, cellulase, β-glucanase, ficin, and sodium chloride is 1: (0.01-0.02): (0.005-0.012): (0.002-0.006): (0.001-0.002).

6. The collagen-containing composition according to claim 1, characterized in that, The preparation method of the modified hyaluronic acid is as follows: (1) Adding hyaluronic acid into an aqueous sodium dodecylbenzenesulfonate solution, stirring evenly, adding 1-ethyl-3-methylimidazolium acetate, and stirring evenly to obtain a mixed solution; (2) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide into the mixed solution, adjusting the pH to 5.4-5.8, reacting at 55-70 °C for 2-6 h, and drying to obtain modified hyaluronic acid.

7. The collagen-containing composition according to claim 6, wherein The mass ratio of the hyaluronic acid, aqueous sodium dodecylbenzenesulfonate solution, and 1-ethyl-3-methylimidazolium acetate is 1: (5-12): (0.05-0.12).

8. The collagen-containing composition according to claim 6, wherein The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the mixed solution is (0.04-0.08): (0.04-0.08):

1.

9. The collagen-containing composition according to claim 6, wherein The mass percentage content of sodium dodecylbenzenesulfonate in the aqueous sodium dodecylbenzenesulfonate solution is 0.5-1.2%.

10. Use of the collagen-containing composition according to any one of claims 1-9 in the preparation of an injection filling material.

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