Biological material for skin and preparation method thereof

Abstract

The invention relates to a biological material for skin and a preparation method thereof, and belongs to the technical field of biology. The invention provides a biological material for skin. The biological material comprises collagen-like protein and sodium hyaluronate, the collagen-like protein and the sodium hyaluronate are firstly crosslinked by the BDDE to form a first network and then crosslinked by the EDC and the NHS to form a second network, or the collagen-like protein and the sodium hyaluronate are firstly crosslinked by the BDDE to form the first network and then crosslinked by the DMTMM to form the second network. According to the biological material, non-animal-sourced pegylated collagen is used for replacing collagen, the collagen has the effects of promoting tissue and cell growth and promoting cell adhesion, meanwhile, certain mechanical performance can be kept, and the pegylated collagen has the advantages of the collagen on the basis that the pegylated collagen has all the advantages of the collagen. And animal-derived viruses and immunogenicity are eliminated fundamentally.

Description

Technical Field

[0001] This invention relates to a biomaterial for skin and its preparation method, belonging to the field of biotechnology. Background Technology

[0002] The skin is divided into three main parts: the epidermis, dermis, and subcutaneous tissue. Artificial dermis is used to replace the dermal layer of the skin and is generally divided into upper and lower layers. The upper layer is a medical silicone rubber membrane with semi-permeable membrane properties, which functions similarly to the epidermis, controlling water evaporation and preventing microbial invasion. The lower layer is a spongy dermal scaffold layer constructed of collagen and chondroitin sulfate, which acts as a scaffold for cell growth, facilitating the invasion and growth of vascular endothelial cells and fibroblasts at the transplant site, forming a scaffold-new capillary-cell complex. After 2-3 weeks of full vascularization, autologous split-thickness skin can be transplanted. Subsequently, the dermal scaffold will gradually degrade and be replaced by newly formed dermal tissue.

[0003] The main indications for artificial dermis include full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, surgical wounds (donor / graft, post-Modular surgery, laser surgery, foot diseases, and wound dehiscence), traumatic wounds (abrasions, lacerations, second-degree burns, and skin avulsions), and drainage wounds. Artificial dermis can effectively guide new dermal regeneration, reduce and inhibit scar hyperplasia, thereby restoring the elasticity and flexibility of the wound surface and improving appearance and function. In some wound repairs, double-layered artificial dermis can even directly cover exposed bone and tendons, replacing traditional flap transplantation. Using artificial dermis on large skin defects allows for early wound closure, reducing the loss of wound fluids, including water and plasma proteins, thus reducing the consumption of nutrients. For wounds after tumor resection, it allows for earlier observation of tumor recurrence, reducing the risk of tumor residue. Meanwhile, after artificial dermal vascularization, only a split-thickness skin graft is needed. Not only is the skin graft survival rate high, but the donor site also heals quickly, with minimal damage and scarring. Compared to flap transplantation surgery, it is shorter and has lower anesthesia risks.

[0004] Currently, commercially available artificial skin products mainly include Integra's double-layer artificial skin and Anika's Hyalomatrix, which use biomaterials as the main components, and Polynovo's NovoSorb BTM, which uses polymeric chemical materials as the main components. Integra's double-layer artificial skin primarily consists of bovine type I collagen and shark chondroitin 6-sulfate. The two components are mixed and precipitated, then cross-linked with glutaraldehyde to form a scaffold. After being freeze-dried and coated with a layer of silica gel, it is stored in phosphate-buffered saline at 2–30°C. Since these materials are all derived from animals, there is a certain risk of animal-derived viruses. In particular, the collagen used is derived from cattle and needs to be imported from New Zealand. The raw material processing is complex, the source is limited, and there is a risk of immunogenicity. Furthermore, the cross-linking agent used is glutaraldehyde, which is highly toxic and poses safety concerns. Anika's Hyalomatrix's main component is benzyl hyaluronic acid, a derivative of hyaluronic acid. Benzyl hyaluronic acid is electrospun into the dermal layer. As a derivative of biomaterials, it has low similarity to the main components of human dermis (which are primarily composed of collagen, elastin, reticular protein, hyaluronic acid, blood vessels, hair follicles, and sweat and sebaceous glands) compared to collagen scaffolds, resulting in poor biocompatibility. Polynovo's NovoSorb BTM's main component is biodegradable polyurethane, which has even lower similarity to the main components of human dermis, leading to even worse biocompatibility. Therefore, there is an urgent need to find artificial dermis that can eliminate the risks of animal-derived viruses and immunogenicity, has high safety, and is as consistent as possible with the composition of human dermis, possessing better biocompatibility to overcome the shortcomings of existing artificial dermis products. Summary of the Invention

[0005] To address the aforementioned problems, the present invention provides a biomaterial for skin, comprising collagen and / or collagen-like proteins; the collagen and / or collagen-like proteins are cross-linked with a cross-linking agent to form a network; the cross-linking agent comprises butylene glycol diglycidyl ether (BDDE), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and / or N-hydroxysuccinimide (NHS).

[0006] In one embodiment of the invention, the biomaterial for skin further includes sodium hyaluronate.

[0007] In one embodiment of the present invention, the biomaterial for skin includes a collagen-like protein and sodium hyaluronate; the collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network; or, the collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to form a second network.

[0008] In one embodiment of the present invention, the method for preparing the biomaterial for skin includes the following steps:

[0009] First dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and / or lyophilized collagen-like substances are dissolved in an alkaline solution and stirred for the first time to obtain a solution; sodium hyaluronate is dissolved in the solution and stirred for the second time to obtain a mixture; butanediol diglycidyl ether is dissolved in the mixture and stirred for the third time to obtain the first cross-linking system;

[0010] First crosslinking step: The first crosslinking system is allowed to stand for the first time to allow the crosslinking reaction to occur, and the first crosslinking product is obtained;

[0011] First freeze-drying step: The first cross-linking product is pre-frozen and then freeze-dried for the first time to obtain a sponge-like freeze-dried product;

[0012] Second dissolution step: Dissolve the spongy lyophilized product in a solvent containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second crosslinking system; or, dissolve the spongy lyophilized product in a solvent containing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to obtain a second crosslinking system;

[0013] Second crosslinking step: The second crosslinking system is allowed to stand for a second time to allow the crosslinking reaction to occur, resulting in the second crosslinking product;

[0014] Second freeze-drying step: After washing the second cross-linking product, a second freeze-drying is performed to obtain a biomaterial for skin use.

[0015] In one embodiment of the present invention, the collagen-like protein is polyethylene glycol-modified collagen-like protein; the collagen-like protein lyophilized powder is polyethylene glycol-modified collagen lyophilized powder.

[0016] In one embodiment of the present invention, in the first dissolution step, the concentration of collagen or collagen-like protein in the dissolution solution is 100-300 mg / mL.

[0017] In one embodiment of the present invention, in the first dissolution step, the concentration of collagen or collagen-like protein in the dissolving solution is 100-120 g / mL.

[0018] In one embodiment of the present invention, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100-300 mg / mL.

[0019] In one embodiment of the present invention, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100-120 g / mL.

[0020] In one embodiment of the present invention, in the first dissolution step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first crosslinking system is 2:1 to 10:1.

[0021] In one embodiment of the present invention, in the first dissolution step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first crosslinking system is 2:1 to 6:1.

[0022] In one embodiment of the present invention, in the first dissolution step, the first stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the rotation speed is 100 to 2000 r / min.

[0023] In one embodiment of the present invention, in the first dissolution step, the first stirring time is 1 to 5 hours, the temperature is 35 to 45°C, and the rotation speed is 400 to 600 r / min.

[0024] In one embodiment of the present invention, in the first dissolution step, the second stirring time is 0.5 to 10 hours, the temperature is 20 to 45°C, and the rotation speed is 100 to 2000 r / min.

[0025] In one embodiment of the present invention, in the first dissolution step, the second stirring time is 1.5 to 10 hours, the temperature is 30 to 45°C, and the rotation speed is 400 to 600 r / min.

[0026] In one embodiment of the present invention, in the first dissolution step, the third stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the rotation speed is 100 to 2000 r / min.

[0027] In one embodiment of the present invention, in the first dissolution step, the third stirring time is 1.5 to 3 hours, the temperature is 30 to 45°C, and the rotation speed is 400 to 1000 r / min.

[0028] In one embodiment of the present invention, the pH of the alkaline solution is 10 to 14.

[0029] In one embodiment of the present invention, the pH of the alkaline solution is 12 to 14.

[0030] In one embodiment of the present invention, the alkaline solution includes sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution and / or sodium carbonate solution.

[0031] In one embodiment of the present invention, the alkaline solution is a sodium hydroxide solution with a pH of 10 to 14.

[0032] In one embodiment of the present invention, the alkaline solution is a sodium hydroxide solution with a pH of 12 to 14.

[0033] In one embodiment of the present invention, in the first crosslinking step, the first standing time is 0.5 to 10 hours and the temperature is 20 to 55°C.

[0034] In one embodiment of the present invention, in the first crosslinking step, the first standing time is 3-4 hours and the temperature is 30-40°C.

[0035] In one embodiment of the present invention, in the first freeze-drying step, the pre-freezing time is 4 to 24 hours and the temperature is -10 to -40°C.

[0036] In one embodiment of the present invention, in the first freeze-drying step, the pre-freezing time is 10 to 14 hours and the temperature is -25 to -30°C.

[0037] In one embodiment of the present invention, in the first freeze-drying step, the first freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

[0038] In one embodiment of the present invention, in the first freeze-drying step, the first freeze-drying time is 14 to 18 hours and the temperature is -20 to -40°C.

[0039] In one embodiment of the present invention, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 10-100 mmol / L and 10-100 mmol / L, respectively.

[0040] In one embodiment of the present invention, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 20-80 mmol / L and 20-80 mmol / L, respectively.

[0041] In one embodiment of the present invention, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 10 to 100 mmol / L.

[0042] In one embodiment of the present invention, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 20-100 mmol / L.

[0043] In one embodiment of the present invention, the solvent includes water and / or an organic solvent; the organic solvent includes ethanol, isopropanol, n-butanol and / or pentanediol.

[0044] In one embodiment of the present invention, the solvent for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is an aqueous ethanol solution with a volume percentage concentration (i.e., volume fraction) of 60-99%; the solvent for 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is water.

[0045] In one embodiment of the present invention, in the second crosslinking step, the second standing time is 12-48 hours and the temperature is 20-40°C.

[0046] In one embodiment of the present invention, in the second crosslinking step, the second standing time is 24-48 hours and the temperature is 20-30°C.

[0047] In one embodiment of the present invention, the washing time in the second freeze-drying step is 1 to 48 hours.

[0048] In one embodiment of the present invention, the washing time in the second freeze-drying step is 4 to 24 hours.

[0049] In one embodiment of the present invention, the cleaning step in the second freeze-drying step includes cleaning with water for injection.

[0050] In one embodiment of the present invention, in the second freeze-drying step, the second freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

[0051] In one embodiment of the present invention, in the second freeze-drying step, the second freeze-drying time is 14 to 18 hours and the temperature is -15 to -40°C.

[0052] In one embodiment of the present invention, the method for preparing the polyethylene glycol-based collagen includes the following steps:

[0053] Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0054] In one embodiment of the present invention, the preparation method of the polyethylene glycol-modified collagen lyophilized powder includes the following steps:

[0055] Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein;

[0056] Freeze-drying step: The reaction product containing polyethylene glycol-modified collagen is freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

[0057] In one embodiment of the present invention, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 4.0 to 10.0 and a temperature of 2 to 40°C for 1 to 48 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0058] In one embodiment of the present invention, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 6.0 to 8.0 and a temperature of 2 to 8°C for 5 to 8 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0059] In one embodiment of the present invention, in the reaction step, the molar ratio of collagen-like protein to polyethylene glycol derivative is 1 to 16:1.

[0060] In one embodiment of the present invention, in the reaction step, the molar ratio of collagen-like protein to polyethylene glycol derivative is 8 to 12:1.

[0061] In one embodiment of the present invention, in the reaction step, the reaction solvent for the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.

[0062] In one embodiment of the present invention, the concentration of the dilute hydrochloric acid solution is 1 to 10 mmol / L; the pH of the dilute hydrochloric acid solution is adjusted to 6.0 to 8.0 using a dilute alkaline solution.

[0063] In one embodiment of the present invention, the dilute alkaline solution is a sodium hydroxide solution or ammonia solution with a pH of 9.0 to 11.0.

[0064] In one embodiment of the present invention, in the reaction step, the concentration of collagen-like protein in the reaction solvent is 1-15 mg / mL.

[0065] In one embodiment of the present invention, in the reaction step, the concentration of collagen-like protein in the reaction solvent is 8-10 mg / mL.

[0066] In one embodiment of the present invention, the freeze-drying step is as follows: the reaction product containing polyethylene glycol-modified collagen and the freeze-drying protectant are mixed and then freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

[0067] In one embodiment of the present invention, in the freeze-drying step, the mass ratio of the reaction product containing polyethylene glycol-based collagen and the freeze-drying protectant is 1:1 to 10.

[0068] In one embodiment of the present invention, the freeze-drying protectant is one or more of mannitol, sucrose, or alanine.

[0069] In one embodiment of the present invention, after the reaction step and before the freeze-drying step, the method further includes a purification step; the purification step is: by filtering, at a temperature of 2 to 8°C, substances with a molecular weight greater than or equal to 30,000 Da in the reaction product containing polyethylene glycol-modified collagen are retained to obtain polyethylene glycol-modified collagen.

[0070] In one embodiment of the present invention, the filtration is dialysis or ultrafiltration.

[0071] In one embodiment of the present invention, the amino acid sequence of the collagen-like protein is shown in SEQ ID NO.1. In SEQ ID NO.1, X is 4Hyp (4-hydroxyproline), that is:

[0072] The amino acid sequence of SEQ ID NO.1 is as follows:

[0073] H-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hy p-Gly-OH

[0074] In one embodiment of the present invention, the polyethylene glycol derivative comprises PEG-40k and PEG-20k.

[0075] In one embodiment of the present invention, the polyethylene glycol derivative is composed of PEG-40k and PEG-20k.

[0076] In one embodiment of the present invention, the molar ratio of PEG-40k to PEG-20k is 0.5 to 6:1.

[0077] In one embodiment of the present invention, the molar ratio of PEG-40k and PEG-20k is 2 to 3:1.

[0078] In one embodiment of the present invention, the number of activating groups of PEG-40k is one or more of eight arms, four arms, two arms or one arm; the number of activating groups of PEG-20k is one or more of eight arms, four arms, two arms or one arm.

[0079] In one embodiment of the present invention, the PEG-40k has four or eight activating groups; the PEG-20k has four or eight activating groups.

[0080] In one embodiment of the present invention, the activating group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; the activating group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.

[0081] In one embodiment of the present invention, the activating group of PEG-40k is -MAL; the activating group of PEG-20k is -MAL.

[0082] In one embodiment of the present invention, one end of the collagen-like protein is attached to a linker peptide; in the reaction step, the polyethylene glycol derivative is modified onto the linker peptide by an activating group to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0083] In one embodiment of the present invention, the linker peptide is attached to the N-terminus of a collagen-like protein.

[0084] In one embodiment of the present invention, the modification site of the polyethylene glycol derivative on the linker peptide is one or more of thiol, amino, carboxyl or imidazole groups.

[0085] In one embodiment of the present invention, the parent nucleus conformation of the polyethylene glycol-based collagen is one or more of HG or TP.

[0086] In one embodiment of the present invention, the parent nucleus conformation of the polyethylene glycol derivative in the polyethylene glycol-based collagen is TP.

[0087] In one embodiment of the present invention, the amino acid configuration of the collagen-like protein in the polyethylene glycol-modified collagen is one or more of the D-type or L-type.

[0088] In one embodiment of the present invention, the molecular weight of the polyethylene glycol-modified collagen is 15,000 to 75,000 Da.

[0089] In one embodiment of the present invention, the molecular weight of the polyethylene glycol-modified collagen is 30,000 to 75,000 Da.

[0090] In one embodiment of the present invention, the biomaterial for skin includes artificial dermis, artificial epidermis, and / or artificial skin.

[0091] In one embodiment of the present invention, the artificial skin includes a combination of dermis and epidermis, a combination of dermis and subcutaneous tissue, and / or a combination of epidermis, dermis and subcutaneous tissue.

[0092] In one embodiment of the present invention, the artificial skin is a functional artificial skin constructed using seed cells.

[0093] In one embodiment of the present invention, the seed cells include immune cells, fibroblasts, vascular endothelial cells, vascular smooth muscle cells and / or hair follicle stem cells; the immune cells include lymphocytes and / or phagocytes.

[0094] The present invention also provides a method for preparing a biomaterial for skin, the method comprising: cross-linking collagen and / or collagen-like proteins with a cross-linking agent to form a network; wherein the cross-linking agent comprises butylene glycol diglycidyl ether, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide, to obtain a biomaterial for skin.

[0095] In one embodiment of the present invention, the method includes: first cross-linking a collagen-like protein and sodium hyaluronate with butylene glycol diglycidyl ether to form a first network, and then cross-linking with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network, thereby obtaining a biomaterial for skin; or, first cross-linking a collagen-like protein and sodium hyaluronate with butylene glycol diglycidyl ether to form a first network, and then cross-linking with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to form a second network, thereby obtaining a biomaterial for skin.

[0096] In one embodiment of the present invention, the method includes the following steps:

[0097] First dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and / or lyophilized collagen-like substances are dissolved in an alkaline solution and stirred for the first time to obtain a solution; sodium hyaluronate is dissolved in the solution and stirred for the second time to obtain a mixture; butanediol diglycidyl ether is dissolved in the mixture and stirred for the third time to obtain the first cross-linking system;

[0098] First crosslinking step: The first crosslinking system is allowed to stand for the first time to allow the crosslinking reaction to occur, and the first crosslinking product is obtained;

[0099] First freeze-drying step: The first cross-linking product is pre-frozen and then freeze-dried for the first time to obtain a sponge-like freeze-dried product;

[0100] Second dissolution step: Dissolve the spongy lyophilized product in a solvent containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second crosslinking system; or, dissolve the spongy lyophilized product in a solvent containing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to obtain a second crosslinking system;

[0101] Second crosslinking step: The second crosslinking system is allowed to stand for a second time to allow the crosslinking reaction to occur, resulting in the second crosslinking product;

[0102] Second freeze-drying step: After washing the second cross-linking product, a second freeze-drying is performed to obtain a biomaterial for skin use.

[0103] In one embodiment of the present invention, the collagen-like protein is polyethylene glycol-modified collagen-like protein; the collagen-like protein lyophilized powder is polyethylene glycol-modified collagen lyophilized powder.

[0104] In one embodiment of the present invention, in the first dissolution step, the concentration of collagen or collagen-like protein in the dissolution solution is 100-300 mg / mL.

[0105] In one embodiment of the present invention, in the first dissolution step, the concentration of collagen or collagen-like protein in the dissolving solution is 100-120 g / mL.

[0106] In one embodiment of the present invention, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100-300 mg / mL.

[0107] In one embodiment of the present invention, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100-120 g / mL.

[0108] In one embodiment of the present invention, in the first dissolution step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first crosslinking system is 2:1 to 10:1.

[0109] In one embodiment of the present invention, in the first dissolution step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first crosslinking system is 2:1 to 6:1.

[0110] In one embodiment of the present invention, in the first dissolution step, the first stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the rotation speed is 100 to 2000 r / min.

[0111] In one embodiment of the present invention, in the first dissolution step, the first stirring time is 1 to 5 hours, the temperature is 35 to 45°C, and the rotation speed is 400 to 600 r / min.

[0112] In one embodiment of the present invention, in the first dissolution step, the second stirring time is 0.5 to 10 hours, the temperature is 20 to 45°C, and the rotation speed is 100 to 2000 r / min.

[0113] In one embodiment of the present invention, in the first dissolution step, the second stirring time is 1.5 to 10 hours, the temperature is 30 to 45°C, and the rotation speed is 400 to 600 r / min.

[0114] In one embodiment of the present invention, in the first dissolution step, the third stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the rotation speed is 100 to 2000 r / min.

[0115] In one embodiment of the present invention, in the first dissolution step, the third stirring time is 1.5 to 3 hours, the temperature is 30 to 45°C, and the rotation speed is 400 to 1000 r / min.

[0116] In one embodiment of the present invention, the pH of the alkaline solution is 10 to 14.

[0117] In one embodiment of the present invention, the pH of the alkaline solution is 12 to 14.

[0118] In one embodiment of the present invention, the alkaline solution includes sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution and / or sodium carbonate solution.

[0119] In one embodiment of the present invention, the alkaline solution is a sodium hydroxide solution with a pH of 10 to 14.

[0120] In one embodiment of the present invention, the alkaline solution is a sodium hydroxide solution with a pH of 12 to 14.

[0121] In one embodiment of the present invention, in the first crosslinking step, the first standing time is 0.5 to 10 hours and the temperature is 20 to 55°C.

[0122] In one embodiment of the present invention, in the first crosslinking step, the first standing time is 3-4 hours and the temperature is 30-40°C.

[0123] In one embodiment of the present invention, in the first freeze-drying step, the pre-freezing time is 4 to 24 hours and the temperature is -10 to -40°C.

[0124] In one embodiment of the present invention, in the first freeze-drying step, the pre-freezing time is 10 to 14 hours and the temperature is -25 to -30°C.

[0125] In one embodiment of the present invention, in the first freeze-drying step, the first freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

[0126] In one embodiment of the present invention, in the first freeze-drying step, the first freeze-drying time is 14 to 18 hours and the temperature is -20 to -40°C.

[0127] In one embodiment of the present invention, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 10-100 mmol / L and 10-100 mmol / L, respectively.

[0128] In one embodiment of the present invention, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 20-80 mmol / L and 20-80 mmol / L, respectively.

[0129] In one embodiment of the present invention, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 10 to 100 mmol / L.

[0130] In one embodiment of the present invention, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 20-100 mmol / L.

[0131] In one embodiment of the present invention, the solvent includes water and / or an organic solvent; the organic solvent includes ethanol, isopropanol, n-butanol and / or pentanediol.

[0132] In one embodiment of the present invention, the solvent for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is an aqueous ethanol solution with a volume percentage concentration (i.e., volume fraction) of 60-99%; the solvent for 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is water.

[0133] In one embodiment of the present invention, in the second crosslinking step, the second standing time is 12-48 hours and the temperature is 20-40°C.

[0134] In one embodiment of the present invention, in the second crosslinking step, the second standing time is 24-48 hours and the temperature is 20-30°C.

[0135] In one embodiment of the present invention, the washing time in the second freeze-drying step is 1 to 48 hours.

[0136] In one embodiment of the present invention, the washing time in the second freeze-drying step is 4 to 24 hours.

[0137] In one embodiment of the present invention, the cleaning step in the second freeze-drying step includes cleaning with water for injection.

[0138] In one embodiment of the present invention, in the second freeze-drying step, the second freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

[0139] In one embodiment of the present invention, in the second freeze-drying step, the second freeze-drying time is 14 to 18 hours and the temperature is -15 to -40°C.

[0140] In one embodiment of the present invention, the method for preparing the polyethylene glycol-based collagen includes the following steps:

[0141] Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0142] In one embodiment of the present invention, the preparation method of the polyethylene glycol-modified collagen lyophilized powder includes the following steps:

[0143] Reaction steps: Collagen-like protein and polyethylene glycol derivative are reacted at pH 4.0–10.0 and temperature 2–40℃ for 1–48 h to obtain a reaction product containing polyethylene glycol-modified collagen-like protein;

[0144] Freeze-drying step: The reaction product containing polyethylene glycol-modified collagen is freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

[0145] In one embodiment of the present invention, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 4.0 to 10.0 and a temperature of 2 to 40°C for 1 to 48 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0146] In one embodiment of the present invention, the reaction step is as follows: reacting collagen-like protein and polyethylene glycol derivative at a pH of 6.0 to 8.0 and a temperature of 2 to 8°C for 5 to 8 hours to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0147] In one embodiment of the present invention, in the reaction step, the molar ratio of collagen-like protein to polyethylene glycol derivative is 1 to 16:1.

[0148] In one embodiment of the present invention, in the reaction step, the molar ratio of collagen-like protein to polyethylene glycol derivative is 8 to 12:1.

[0149] In one embodiment of the present invention, in the reaction step, the reaction solvent for the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.

[0150] In one embodiment of the present invention, the concentration of the dilute hydrochloric acid solution is 1 to 10 mmol / L; the pH of the dilute hydrochloric acid solution is adjusted to 6.0 to 8.0 using a dilute alkaline solution.

[0151] In one embodiment of the present invention, the dilute alkaline solution is a sodium hydroxide solution or ammonia solution with a pH of 9.0 to 11.0.

[0152] In one embodiment of the present invention, in the reaction step, the concentration of collagen-like protein in the reaction solvent is 1-15 mg / mL.

[0153] In one embodiment of the present invention, in the reaction step, the concentration of collagen-like protein in the reaction solvent is 8-10 mg / mL.

[0154] In one embodiment of the present invention, the freeze-drying step is as follows: the reaction product containing polyethylene glycol-modified collagen and the freeze-drying protectant are mixed and then freeze-dried to obtain polyethylene glycol-modified collagen freeze-dried powder.

[0155] In one embodiment of the present invention, in the freeze-drying step, the mass ratio of the reaction product containing polyethylene glycol-based collagen and the freeze-drying protectant is 1:1 to 10.

[0156] In one embodiment of the present invention, the freeze-drying protectant is one or more of mannitol, sucrose, or alanine.

[0157] In one embodiment of the present invention, after the reaction step and before the freeze-drying step, the method further includes a purification step; the purification step is: by filtering, at a temperature of 2 to 8°C, substances with a molecular weight greater than or equal to 30,000 Da in the reaction product containing polyethylene glycol-modified collagen are retained to obtain polyethylene glycol-modified collagen.

[0158] In one embodiment of the present invention, the filtration is dialysis or ultrafiltration.

[0159] In one embodiment of the present invention, the amino acid sequence of the collagen-like protein is shown in SEQ ID NO.1. In SEQ ID NO.1, X is 4Hyp (4-hydroxyproline), that is:

[0160] The amino acid sequence of SEQ ID NO.1 is as follows:

[0161] H-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Lys-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hyp-Gly-Asp-Hy p-Gly-OH

[0162] In one embodiment of the present invention, the polyethylene glycol derivative comprises PEG-40k and PEG-20k.

[0163] In one embodiment of the present invention, the polyethylene glycol derivative is composed of PEG-40k and PEG-20k.

[0164] In one embodiment of the present invention, the molar ratio of PEG-40k to PEG-20k is 0.5 to 6:1.

[0165] In one embodiment of the present invention, the molar ratio of PEG-40k and PEG-20k is 2 to 3:1.

[0166] In one embodiment of the present invention, the number of activating groups of PEG-40k is one or more of eight arms, four arms, two arms or one arm; the number of activating groups of PEG-20k is one or more of eight arms, four arms, two arms or one arm.

[0167] In one embodiment of the present invention, the PEG-40k has four or eight activating groups; the PEG-20k has four or eight activating groups.

[0168] In one embodiment of the present invention, the activating group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; the activating group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.

[0169] In one embodiment of the present invention, the activating group of PEG-40k is -MAL; the activating group of PEG-20k is -MAL.

[0170] In one embodiment of the present invention, one end of the collagen-like protein is attached to a linker peptide; in the reaction step, the polyethylene glycol derivative is modified onto the linker peptide by an activating group to obtain a reaction product containing polyethylene glycol-modified collagen-like protein.

[0171] In one embodiment of the present invention, the linker peptide is attached to the N-terminus of a collagen-like protein.

[0172] In one embodiment of the present invention, the modification site of the polyethylene glycol derivative on the linker peptide is one or more of thiol, amino, carboxyl or imidazole groups.

[0173] In one embodiment of the present invention, the parent nucleus conformation of the polyethylene glycol-based collagen is one or more of HG or TP.

[0174] In one embodiment of the present invention, the parent nucleus conformation of the polyethylene glycol derivative in the polyethylene glycol-based collagen is TP.

[0175] In one embodiment of the present invention, the amino acid configuration of the collagen-like protein in the polyethylene glycol-modified collagen is one or more of the D-type or L-type.

[0176] In one embodiment of the present invention, the molecular weight of the polyethylene glycol-modified collagen is 15,000 to 75,000 Da.

[0177] In one embodiment of the present invention, the molecular weight of the polyethylene glycol-modified collagen is 30,000 to 75,000 Da.

[0178] In one embodiment of the present invention, the biomaterial for skin includes artificial dermis, artificial epidermis, and / or artificial skin.

[0179] In one embodiment of the present invention, the artificial skin includes a combination of dermis and epidermis, a combination of dermis and subcutaneous tissue, and / or a combination of epidermis, dermis and subcutaneous tissue.

[0180] In one embodiment of the present invention, the artificial skin is a functional artificial skin constructed using seed cells.

[0181] In one embodiment of the present invention, the seed cells include immune cells, fibroblasts, vascular endothelial cells, vascular smooth muscle cells and / or hair follicle stem cells; the immune cells include lymphocytes and / or phagocytes.

[0182] The technical solution of this invention has the following advantages:

[0183] This invention provides a biomaterial for skin, comprising a collagen-like protein and sodium hyaluronate. The collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network; alternatively, the collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to form a second network. The biomaterial for skin has the following advantages:

[0184] First, non-animal-derived polyethylene glycol-based collagen is used to replace collagen. Collagen promotes tissue and cell growth and cell adhesion, while maintaining certain mechanical properties. In addition to having all the advantages of collagen, polyethylene glycol-based collagen also fundamentally eliminates animal-derived viruses and immunogenicity.

[0185] Secondly, the addition of sodium hyaluronate, which is naturally present in the skin, to polyethylene glycol-modified collagen creates a combination of components that are more consistent with the composition of human dermis, resulting in better biocompatibility. Furthermore, hyaluronic acid participates in every step of wound repair and has non-specific effects on cell receptors (CD44 / RHAMM and ICAM-1), effectively promoting tissue growth and repair.

[0186] Third, the cross-linking agents used (butanediol diglycidyl ether, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide) have no potential cytotoxicity and are highly safe.

[0187] Fourth, it has pores suitable for cell growth, which can promote the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes.

[0188] Therefore, the biomaterials used for skin can serve as artificial dermis, artificial epidermis, or artificial skin, and have broad application prospects in the fields of skin regeneration and tissue repair.

[0189] Further, the preparation method of the biomaterial for skin includes a first dissolution step, a first cross-linking step, a first freeze-drying step, a second dissolution step, a second cross-linking step, and a second freeze-drying step; wherein, the first dissolution step is as follows: collagen, collagen-like substances, collagen freeze-dried powder, and / or collagen-like freeze-dried powder are dissolved in an alkaline solution and stirred for the first time to obtain a solution; sodium hyaluronate is dissolved in the solution and stirred for the second time to obtain a mixture; butylene glycol diglycidyl ether is dissolved in the mixture and stirred for the third time to obtain a first cross-linking system; the first cross-linking step is as follows: the first cross-linking system is allowed to stand for the first time to undergo a cross-linking reaction to obtain a first cross-linking product; the first freeze-drying step... The steps are as follows: First, the first cross-linking product is pre-frozen and then freeze-dried for the first time to obtain a sponge-like freeze-dried product. Second, the sponge-like freeze-dried product is dissolved in a solvent containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second cross-linking system; alternatively, the sponge-like freeze-dried product is dissolved in a solvent containing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to obtain a second cross-linking system. Second, the second cross-linking system is subjected to a second standing period to allow the cross-linking reaction to occur, yielding a second cross-linking product. Second, the second cross-linking product is washed and then freeze-dried a second time to obtain a biomaterial for skin. The biomaterial prepared using this method promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus aiding in skin tissue growth and repair.

[0190] Furthermore, the collagen-like protein is a polyethylene glycol-modified collagen-like protein; the collagen-like protein lyophilized powder is a polyethylene glycol-modified collagen-like protein lyophilized powder. Under this configuration, the biomaterials for skin preparation exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0191] Furthermore, in the first dissolution step, the concentration of collagen or collagen-like proteins in the dissolving solution is 100–300 mg / mL. Under this setting, biomaterials suitable for skin application can be successfully prepared.

[0192] Furthermore, in the first dissolution step, the concentration of collagen or collagen-like protein in the dissolving solution is 100–120 g / mL. Under this setting, the biomaterials prepared for skin use exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0193] Furthermore, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100–300 mg / mL. Under this setting, biomaterials suitable for skin application can be successfully prepared.

[0194] Furthermore, in the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100–120 g / mL. Under this setting, the biomaterial prepared for skin has a better effect on promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0195] Furthermore, in the first dissolution step, the molar ratio of butylene glycol diglycidyl ether to sodium hyaluronate in the first crosslinking system is 2:1 to 10:1. Under this setting, biomaterials for skin application can be successfully prepared.

[0196] Furthermore, in the first dissolution step, the molar ratio of butylene glycol diglycidyl ether to sodium hyaluronate in the first crosslinking system is 2:1 to 6:1. Under this setting, the biomaterials prepared for skin exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0197] Furthermore, in the first dissolution step, the first stirring time is 0.5–10 hours, the temperature is 20–65°C, and the rotation speed is 100–2000 r / min. Under these settings, biomaterials for skin application can be successfully prepared.

[0198] Furthermore, in the first dissolution step, the first stirring time is 1–5 hours, the temperature is 40–45°C, and the rotation speed is 400–600 r / min. Under these conditions, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0199] Furthermore, in the first dissolution step, the second stirring time is 0.5–10 hours, the temperature is 20–45°C, and the rotation speed is 100–2000 r / min. Under these settings, biomaterials for skin application can be successfully prepared.

[0200] Furthermore, in the first dissolution step, the second stirring time is 1.5–10 hours, the temperature is 30–45°C, and the stirring speed is 400–600 r / min. Under these conditions, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0201] Furthermore, in the first dissolution step, the third stirring time is 0.5–10 hours, the temperature is 20–65°C, and the rotation speed is 100–2000 r / min. Under these settings, biomaterials for skin application can be successfully prepared.

[0202] Furthermore, in the first dissolution step, the third stirring time is 1.5–3 hours, the temperature is 30–45°C, and the stirring speed is 400–1000 r / min. Under these conditions, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0203] Furthermore, the pH of the alkaline solution is 10–14. Under this setting, biomaterials for skin application can be successfully prepared.

[0204] Furthermore, the pH of the alkaline solution is 12-14. Under this setting, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0205] Furthermore, in the first cross-linking step, the first resting time is 0.5–10 hours, and the temperature is 20–55°C. Under these conditions, biomaterials for skin application can be successfully prepared.

[0206] Furthermore, in the first cross-linking step, the first resting time is 3-4 hours and the temperature is 30-40°C. Under this setting, the biomaterials prepared for skin have a better effect on promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0207] Furthermore, in the first freeze-drying step, the pre-freezing time is 4–24 hours and the temperature is -10 to -40°C. Under this setting, biomaterials for skin application can be successfully prepared.

[0208] Furthermore, in the first freeze-drying step, the pre-freezing time is 10–14 hours and the temperature is -25–-30°C. Under this setting, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0209] Furthermore, in the first freeze-drying step, the freeze-drying time is 12–24 hours and the temperature is -15 to -40°C. Under this setting, biomaterials for skin application can be successfully prepared.

[0210] Furthermore, in the first freeze-drying step, the freeze-drying time is 14–18 hours and the temperature is -20 to -40°C. Under this setting, the biomaterials prepared for skin use exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0211] Furthermore, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 10–100 mmol / L and 10–100 mmol / L, respectively. Under this setting, biomaterials molded for skin application can be successfully prepared.

[0212] Furthermore, in the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 20–80 mmol / L and 20–80 mmol / L, respectively. Under this setting, the biomaterials prepared for skin exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0213] Furthermore, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 10–100 mmol / L. Under this setting, biomaterials molded for skin application can be successfully prepared.

[0214] Furthermore, in the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 20–100 mmol / L. Under this setting, the biomaterials prepared for skin exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0215] Furthermore, in the second cross-linking step, the second settling time is 12–48 hours, and the temperature is 20–40°C. Under these conditions, biomaterials for skin application can be successfully prepared.

[0216] Furthermore, in the second cross-linking step, the second settling time is 24–48 hours, and the temperature is 20–30°C. Under this setting, the biomaterials prepared for skin have a better effect on promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0217] Furthermore, in the second freeze-drying step, the washing time is 1–48 hours. Under this setup, biomaterials suitable for skin application can be successfully prepared.

[0218] Furthermore, in the second freeze-drying step, the washing time is 4–24 hours. Under this setting, the resulting biomaterials for skin exhibit better growth-promoting effects on skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair.

[0219] Furthermore, in the second freeze-drying step, the freeze-drying time is 12–24 hours and the temperature is -15 to -40°C. Under this setting, biomaterials for skin application can be successfully prepared.

[0220] Furthermore, in the second freeze-drying step, the freeze-drying time is 14–18 hours and the temperature is -15 to -40°C. Under this setting, the biomaterials prepared for skin application exhibit better effects in promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, as well as immune cells such as lymphocytes, thus contributing to skin tissue growth and repair. Attached Figure Description

[0221] Figure 1 Environmental scanning electron microscope image of artificial dermis 1.

[0222] Figure 2 Cell survival rate after culturing different cells with artificial dermis. Detailed Implementation

[0223] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0224] Unless otherwise specified, the experimental steps or conditions described in the following examples can be performed according to the conventional experimental procedures and conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. The synthesis of collagen-like proteins and the connection between collagen-like proteins and linking peptides in the following examples were performed by Shanghai Ambio Biotechnology Co., Ltd. (AmbioPharm.InC.). The PEG derivatives used in the following examples were all purchased from Xiamen Sinobond Biotechnology Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and butanediol diglycidyl ether (BDDE) used in the following examples were purchased from SIGMA; sodium hyaluronate used in the following examples was purchased from Bloomage Biotechnology Co., Ltd.; the pre-freezing and lyophilization in the following examples were performed using an FD-1A-50 lyophilizer purchased from Shanghai Jipu Electronic Technology Co., Ltd.; and the stirring in the following examples was performed using a magnetic stirrer purchased from Shanghai Titan Technology Co., Ltd.

[0225] Example 1: An artificial dermis and its preparation

[0226] This embodiment provides an artificial dermis, the preparation method of which includes the following steps:

[0227] First dissolution step: Polyethylene glycol-modified collagen lyophilized powder (for details on the preparation of polyethylene glycol-modified collagen lyophilized powder, please refer to Example 1 of patent application CN115990969A) was dissolved in a sodium hydroxide solution (solvent: water) at pH 12, ensuring the concentration of polyethylene glycol-modified collagen in the sodium hydroxide solution was 100 mg / mL. The solution was then stirred for the first time at 40°C and 400 r / min for 2 hours to obtain the dissolved product. Sodium hyaluronate was dissolved in a solvent to achieve a concentration of 100 mg / mL. The solution was then stirred for a second time at 40°C and 400 rpm for 2 hours to obtain a mixed solution. Butylene glycol diglycidyl ether was dissolved in the mixed solution to achieve a molar ratio of 4:1 between the butylene glycol diglycidyl ether and sodium hyaluronate. The solution was then stirred for a third time at 40°C and 400 rpm for 2 hours to obtain the first crosslinking system.

[0228] First crosslinking step: The first crosslinking system is allowed to stand at 40°C for 3 hours to allow the crosslinking reaction to occur, and the first crosslinking product is obtained;

[0229] First freeze-drying step: Pour the first cross-linking product into a freeze-drying pan, pre-freeze at -25℃ for 12h, and then freeze-dry for 16h at -25℃ and vacuum degree (75Pa) to obtain a sponge-like freeze-dried product.

[0230] Second dissolution step: The spongy lyophilized product was dissolved in a 95% (v / v) aqueous ethanol solution containing 60 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mmol / L N-hydroxysuccinimide, so that the spongy lyophilized product was immersed in the 95% (v / v) aqueous ethanol solution to obtain the second crosslinking system;

[0231] Second crosslinking step: The second crosslinking system is subjected to a second standing at 25°C for 24 hours to allow the crosslinking reaction to occur, thereby obtaining the second crosslinking product;

[0232] Second freeze-drying step: After washing the second cross-linked product in water for injection for 12 hours, freeze-dry it for 16 hours at a temperature of -25℃ and a vacuum of 75Pa to obtain artificial dermis (this artificial dermis is named artificial dermis 1).

[0233] Example 2: An artificial dermis and its preparation

[0234] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the concentration of polyethylene glycol-modified collagen (100 mg / mL) in the first dissolution step is replaced with: 20 mg / mL, 40 mg / mL, 80 mg / mL, 120 mg / mL, 200 mg / mL, and 300 mg / mL respectively.

[0235] The above-mentioned artificial dermis are named Artificial Dermis 2 to 7 in sequence.

[0236] Example 3: An artificial dermis and its preparation

[0237] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the concentration of sodium hyaluronate (100 mg / mL) in the first dissolution step is replaced with: 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL, 120 mg / mL, 200 mg / mL, and 300 mg / mL respectively.

[0238] The above-mentioned artificial dermis are named Artificial Dermis 8 to 14 in sequence.

[0239] Example 4: An artificial dermis and its preparation

[0240] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, except that the molar ratio (4:1) of butylene glycol diglycidyl ether and sodium hyaluronate in the first dissolution step is replaced with: 0.5:1, 1:1, 2:1, 6:1, 8:1, 10:1 respectively.

[0241] The above-mentioned artificial dermis are named Artificial Dermis 15 to 20 in sequence.

[0242] Example 5: An artificial dermis and its preparation

[0243] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the first stirring time (2h) in the first dissolution step is replaced with: 0.5h, 1h, 1.5h, 3h, 5h, and 10h respectively.

[0244] The aforementioned artificial dermis are named Artificial Dermis 21 to 26 in sequence.

[0245] Example 6: An artificial dermis and its preparation

[0246] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the first stirring temperature (40°C) in the first dissolution step is replaced with: 20°C, 25°C, 30°C, 35°C, 45°C, 55°C, and 65°C respectively.

[0247] The above-mentioned artificial dermis are named Artificial Dermis 27 to 33 in sequence.

[0248] Example 7: An artificial dermis and its preparation

[0249] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the first stirring speed (400 r / min) in the first dissolution step is replaced with: 100 r / min, 200 r / min, 600 r / min, 1000 r / min, and 2000 r / min respectively.

[0250] The above-mentioned artificial dermis are named Artificial Dermis 34 to 38 in sequence.

[0251] Example 8: An artificial dermis and its preparation

[0252] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the second stirring time (2h) in the first dissolution step is replaced with: 0.5h, 1h, 1.5h, 3h, 5h, and 10h respectively.

[0253] The above-mentioned artificial dermis are named Artificial Dermis 39 to 44 in sequence.

[0254] Example 9: An artificial dermis and its preparation

[0255] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the pH (12) of the sodium hydroxide solution in the first dissolution step is replaced with: 7, 8, 9, 10, 11, 13, 14 respectively.

[0256] The above-mentioned artificial dermis are named artificial dermis 45 to 51 in sequence.

[0257] Example 10: An artificial dermis and its preparation

[0258] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the second stirring temperature (40°C) in the first dissolution step is replaced with: 20°C, 25°C, 30°C, 35°C, 45°C, 55°C, and 65°C respectively.

[0259] The above-mentioned artificial dermis are named Artificial Dermis 52 to 58 in sequence.

[0260] Example 11: An artificial dermis and its preparation

[0261] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the second stirring speed (400 r / min) in the first dissolution step is replaced with: 100 r / min, 200 r / min, 600 r / min, 1000 r / min, and 2000 r / min respectively.

[0262] The above-mentioned artificial dermis are named Artificial Dermis 59 to 63 in sequence.

[0263] Example 12: An artificial dermis and its preparation

[0264] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the third stirring time (2h) in the first dissolution step is replaced with: 0.5h, 1h, 1.5h, 3h, 5h, and 10h respectively.

[0265] The above-mentioned artificial dermis are named Artificial Dermis 64 to 69 in sequence.

[0266] Example 13: An artificial dermis and its preparation

[0267] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the third stirring temperature (40°C) in the first dissolution step is replaced with: 20°C, 25°C, 30°C, 35°C, 45°C, 55°C, and 65°C respectively.

[0268] The above-mentioned artificial dermis are named Artificial Dermis 70 to 76 in sequence.

[0269] Example 14: An artificial dermis and its preparation

[0270] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the third stirring speed (400 r / min) in the first dissolution step is replaced with: 100 r / min, 200 r / min, 600 r / min, 1000 r / min, and 2000 r / min respectively.

[0271] The above-mentioned artificial dermis are named Artificial Dermis 77-81 in sequence.

[0272] Example 15: An artificial dermis and its preparation

[0273] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the first resting time (3h) in the first crosslinking step is replaced with: 0.5h, 1h, 2h, 4h, 5h, 10h respectively.

[0274] The above-mentioned artificial dermis are named Artificial Dermis 82 to 87 in sequence.

[0275] Example 16: An artificial dermis and its preparation

[0276] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the first standing temperature (40°C) in the first crosslinking step is replaced with 25°C, 30°C, 35°C, 45°C, and 55°C respectively.

[0277] The above-mentioned artificial dermis are named Artificial Dermis 88 to 92 in sequence.

[0278] Example 17: An artificial dermis and its preparation

[0279] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Example 1, except that the second resting time (24h) in the second crosslinking step is replaced with: 6h, 8h, 12h, 36h, and 48h respectively.

[0280] The above-mentioned artificial dermis are named Artificial Dermis 93 to 97 in sequence.

[0281] Example 18: An artificial dermis and its preparation

[0282] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, except that the second standing temperature (25°C) in the second crosslinking step is replaced with: 4°C, 20°C, 30°C, 35°C, and 40°C respectively.

[0283] The above-mentioned artificial dermis are named Artificial Dermis 98 to 102 in sequence.

[0284] Example 19: An artificial dermis and its preparation

[0285] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the pre-freezing time (12h) in the first freeze-drying step is replaced with: 4h, 8h, 18h, and 24h respectively.

[0286] The above-mentioned artificial dermis are named artificial dermis 103 to 106 in sequence.

[0287] Example 20: An artificial dermis and its preparation

[0288] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the pre-freezing temperature (-25℃) in the first freeze-drying step is replaced with: -10℃, -15℃, -20℃, -30℃, -35℃, and -40℃ respectively.

[0289] The above-mentioned artificial dermis are named Artificial Dermis 107 to 112 in sequence.

[0290] Example 21: An artificial dermis and its preparation

[0291] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the first freeze-drying time (16h) in the first freeze-drying step is replaced with: 6h, 8h, 10h, 14h, 18h, 24h respectively.

[0292] The above-mentioned artificial dermis are named Artificial Dermis 113 to 118 in sequence.

[0293] Example 22: An artificial dermis and its preparation

[0294] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the first freeze-drying temperature (-25℃) in the first freeze-drying step is replaced with: -10℃, -15℃, -20℃, -30℃, -35℃, and -40℃ respectively.

[0295] The above-mentioned artificial dermis are named Artificial Dermis 119 to 124 in sequence.

[0296] Example 23: An artificial dermis and its preparation

[0297] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the second freeze-drying time (16h) in the second freeze-drying step is replaced with: 6h, 8h, 10h, 14h, 18h, 24h respectively.

[0298] The above-mentioned artificial dermis are named Artificial Dermis 125-130 in sequence.

[0299] Example 24: An artificial dermis and its preparation

[0300] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the second freeze-drying temperature (-25℃) in the second freeze-drying step is replaced with: -10℃, -15℃, -20℃, -30℃, -35℃, and -40℃ respectively.

[0301] The aforementioned artificial dermis are named Artificial Dermis 131 to 136 in sequence.

[0302] Example 25: An artificial dermis and its preparation

[0303] This embodiment provides an artificial dermis, which is based on the artificial dermis 1 in Embodiment 1, except that the washing time (12h) in the second freeze-drying step is replaced with: 4h, 6h, 8h, 14h, 16h, and 24h respectively.

[0304] The above-mentioned artificial dermis are named Artificial Dermis 137 to 142 in sequence.

[0305] Example 26: An artificial dermis and its preparation

[0306] This embodiment provides an artificial dermis, which is based on artificial dermis 1 in Example 1, except that the 95% (v / v) aqueous ethanol solution containing 60 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mmol / L N-hydroxysuccinimide in the second dissolution step is replaced with:

[0307] A 95% (v / v) aqueous solution of ethanol containing 10 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mmol / L N-hydroxysuccinimide;

[0308] A 95% (v / v) aqueous solution of ethanol containing 20 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 20 mmol / L N-hydroxysuccinimide;

[0309] A 95% (v / v) aqueous solution of ethanol containing 40 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mmol / L N-hydroxysuccinimide;

[0310] A 95% (v / v) aqueous solution of ethanol containing 80 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 80 mmol / L N-hydroxysuccinimide;

[0311] A 95% (v / v) aqueous solution of ethanol containing 100 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mmol / L N-hydroxysuccinimide.

[0312] The above-mentioned artificial dermis are named Artificial Dermis 143 to 147 in sequence.

[0313] Example 27: An artificial dermis and its preparation

[0314] This embodiment provides an artificial dermis, which is based on artificial dermis 1 in Example 1, except that the 95% (v / v) aqueous ethanol solution containing 60 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mmol / L N-hydroxysuccinimide in the second dissolution step is replaced with:

[0315] A 10 mmol / L aqueous solution of DMTMN;

[0316] A 20 mmol / L aqueous solution of DMTMN;

[0317] A 40 mmol / L aqueous solution of DMTMN;

[0318] A 60 mmol / L aqueous solution of DMTMN;

[0319] An aqueous solution of DMTMN with a concentration of 80 mmol / L;

[0320] A 100 mmol / L aqueous solution of DMTMN.

[0321] The above-mentioned artificial dermis are named Artificial Dermis 148 to 153 in sequence.

[0322] Comparative Example 1: An artificial dermis and its preparation

[0323] This comparative example provides an artificial dermis, which is a product of Integra. According to the literature "I VYannas, E Lee. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. [J] Proc Natl Acad Sci USA. 1989 Feb; 86(3): 933-7.", its collagen and chondroitin sulfate artificial scaffold has a pore size of 20-125 μm, and the autologous dermal cells and epidermal cells seeded in it have morphological activity. Its main materials are bovine type I collagen and chondroitin 6-sulfate. The manufacturing method is described in US Patent US4060081A.

[0324] Experiment Example 1: The Influence of Preparation Process on the Properties of Artificial Dermal Skin

[0325] This experimental example demonstrates the effect of the preparation process on the properties of artificial dermis. The experimental procedure is as follows:

[0326] Experiment 1: Following the detection method in the literature "Fergal J. O'Brien, Brendan A. Harley, Ioannis V. Yannas, Lorna Gibson, Influence of freezing rate on pore structure in freeze-dried colloidal-GAG scaffolds, Biomaterials, Volume 25, Issue 6, 2004, Pages 1077-1086, ISSN 0142-9612," an environmental scanning electron microscope (Thermo Fisher Scientific QUANTA) was used. 200) The pore size of artificial dermis 1-68 was measured. To ensure the accurate measurement of the pore size of the artificial dermis under wet conditions, the artificial dermis was first cut into 1cm×1cm pieces, and then immersed in PBS buffer (prepared by adding 0.00144g / 100mL KH2PO4 and 0.01062g / 100mL Na2HPO4 to 0.9g / 100mL sodium chloride solution, and adjusting the pH to 7.4 with 0.1M sodium hydroxide) for 20 minutes. Finally, the artificial dermis was fixed with conductive adhesive without dripping water. The electron microscope was used at 800x magnification, and the pore size was measured using an Image J scale bar. The measurement results are shown in Table 1 and 200. Figure 1 .

[0327] Experiment 2: In vitro cell assays were performed on artificial dermis types 1, 17, and 51 to detect their cytotoxicity, cell proliferation capacity, and cell adhesion ability. The specific assay methods are as follows:

[0328] Artificial dermis was cut into 1 cm diameter circles and then immersed in PBS buffer for 20 minutes to obtain the test sample. A blank control without artificial dermis was used. Cells (human fibroblasts and vascular endothelial cells, purchased from Changzhou Huayang Chemical Co., Ltd.) were added at 2.5 × 10⁻⁶ cells / mL. 5 After inoculating each well of a 24-well plate with 2 mL of complete culture medium (purchased from Changzhou Huayang Chemical Co., Ltd.) at a seeding rate of 1 cell / well, the test samples were added to the 24-well plates. After inoculation, the 24-well plates were placed in a cell culture incubator at 37°C and 5% (v / v) CO2. After 1 day (for cytotoxicity testing) and 5 days (for cell proliferation testing), the 24-well plates were removed from the cell culture incubator, and the test samples were removed and transferred to new 24-well plates. After transfer, 0.5 mL of 1 mg / mL MTT solution (purchased from Changzhou Huayang Chemical Co., Ltd.) was added to each well. The sample was added to each well of a 24-well plate containing the test sample. After addition, the 24-well plate was incubated at 37°C and 5% (v / v) CO2 in a cell culture incubator. After 2 hours of incubation, the 24-well plate was removed from the cell culture incubator, and 1 mL of isopropanol was added to each well to dissolve the sample. After addition, the solution in the 24-well plate was mixed with a shaker, and then the 24-well plate was placed in a microplate reader. The absorbance of each well was measured at 570 nm (after subtracting the background of the blank group without cell growth). The cell viability after culture of different cells and artificial dermis was calculated based on the measurement results. The measurement results are shown in Tables 2 and 3, and the calculation results are shown in Tables 2 and 3. Figure 2 The formula for calculating cell survival rate is: Cell survival rate % = 100% × (OD sample group - OD blank group) / OD blank group.

[0329] From Table 1 and Figure 1It is known that artificial dermal scaffolds have pore sizes ranging from 11 to 104 μm. According to the literature "I VYannas, E Lee. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.[J]Proc Natl AcadSci USA.1989 Feb;86(3):933-7.", the suitable pore size for the growth of fibroblasts and vascular endothelial cells is 20 to 125 μm. Some embodiments conform to the optimal pore size range, so cells can infiltrate into the scaffold and proliferate.

[0330] From Tables 2-3 and Figure 2 It was found that vascular endothelial cells and fibroblasts proliferated 5 days after inoculation with artificial dermis, and the artificial dermis group showed greater cell proliferation compared to the control (culture medium + cells). This result indicates that artificial dermis is suitable for cell growth, and cells can proliferate and grow after being infiltrated into the artificial dermis scaffold.

[0331] Table 1. Average pore size of artificial dermis 1–153

[0332]

[0333]

[0334]

[0335]

[0336] Table 2. Increase in absorbance after different time periods of vascular endothelial cell inoculation into artificial dermis culture.

[0337]

[0338] Table 3. Increase in absorbance after different time periods of human fibroblast culture inoculated into artificial dermis.

[0339]

[0340] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A biomaterial for use on the skin, characterized in that, The biomaterials for skin include collagen and / or collagen-like proteins; the collagen and / or collagen-like proteins are cross-linked with a cross-linking agent to form a network; the cross-linking agent includes butylene glycol diglycidyl ether, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

2. The biomaterial as described in claim 1, characterized in that, The biomaterials used for the skin also include sodium hyaluronate.

3. The biomaterial as described in claim 1 or 2, characterized in that, The biomaterial for skin includes collagen-like protein and sodium hyaluronate; the collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network, or the collagen-like protein and sodium hyaluronate are first cross-linked with butylene glycol diglycidyl ether to form a first network, and then cross-linked with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to form a second network.

4. The biomaterial according to any one of claims 1 to 3, characterized in that, The method for preparing the biomaterial for skin includes the following steps: First dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and / or lyophilized collagen-like substances are dissolved in an alkaline solution and stirred for the first time to obtain a solution; sodium hyaluronate is dissolved in the solution and stirred for the second time to obtain a mixture; butanediol diglycidyl ether is dissolved in the mixture and stirred for the third time to obtain the first cross-linking system; First crosslinking step: The first crosslinking system is allowed to stand for the first time to allow the crosslinking reaction to occur, and the first crosslinking product is obtained; First freeze-drying step: The first cross-linking product is pre-frozen and then freeze-dried for the first time to obtain a sponge-like freeze-dried product; Second dissolution step: Dissolve the spongy lyophilized product in a solvent containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second crosslinking system; or, dissolve the spongy lyophilized product in a solvent containing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to obtain a second crosslinking system; Second crosslinking step: The second crosslinking system is allowed to stand for a second time to allow the crosslinking reaction to occur, resulting in the second crosslinking product; Second freeze-drying step: After washing the second cross-linking product, a second freeze-drying is performed to obtain a biomaterial for skin use.

5. The biomaterial as described in claim 4, characterized in that, The collagen-like protein is polyethylene glycol-modified collagen; the collagen-like protein freeze-dried powder is polyethylene glycol-modified collagen freeze-dried powder.

6. The biomaterial as described in claim 4 or 5, characterized in that, In the first dissolution step, the concentration of collagen or collagen-like protein in the dissolution solution is 100–300 mg / mL.

7. The biomaterial according to any one of claims 4 to 6, characterized in that, In the first dissolution step, the concentration of sodium hyaluronate in the mixture is 100–300 mg / mL.

8. The biomaterial according to any one of claims 4 to 7, characterized in that, In the first dissolution step, the molar ratio of butanediol diglycidyl ether to sodium hyaluronate in the first crosslinking system is 2:1 to 10:

1.

9. The biomaterial according to any one of claims 4 to 8, characterized in that, In the first dissolution step, the first stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the rotation speed is 100 to 2000 r / min.

10. The biomaterial according to any one of claims 4 to 9, characterized in that, In the first dissolution step, the second stirring time is 0.5 to 10 hours, the temperature is 20 to 45°C, and the rotation speed is 100 to 2000 r / min.

11. The biomaterial according to any one of claims 4 to 10, characterized in that, In the first dissolution step, the third stirring time is 0.5 to 10 hours, the temperature is 20 to 65°C, and the stirring speed is 100 to 2000 r / min.

12. The biomaterial according to any one of claims 4 to 11, characterized in that, The pH of the alkaline solution is 10–14.

13. The biomaterial according to any one of claims 4 to 12, characterized in that, In the first crosslinking step, the first standing time is 0.5 to 10 hours and the temperature is 20 to 55°C.

14. The biomaterial according to any one of claims 4 to 13, characterized in that, In the first freeze-drying step, the pre-freezing time is 4 to 24 hours and the temperature is -10 to -40°C.

15. The biomaterial according to any one of claims 4 to 14, characterized in that, In the first freeze-drying step, the freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

16. The biomaterial according to any one of claims 4 to 15, characterized in that, In the second dissolution step, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the solvent are 10–100 mmol / L and 10–100 mmol / L, respectively.

17. The biomaterial according to any one of claims 4 to 16, characterized in that, In the second dissolution step, the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride in the solvent is 10-100 mmol / L.

18. The biomaterial according to any one of claims 4 to 17, characterized in that, In the second crosslinking step, the second standing time is 12-48 hours and the temperature is 20-40°C.

19. The biomaterial according to any one of claims 4 to 18, characterized in that, In the second freeze-drying step, the washing time is 1 to 48 hours.

20. The biomaterial according to any one of claims 4 to 19, characterized in that, In the second freeze-drying step, the freeze-drying time is 12 to 24 hours and the temperature is -15 to -40°C.

21. A method for preparing a biomaterial for skin, characterized in that, The method includes: cross-linking collagen and / or collagen-like proteins with a cross-linking agent to form a network, thereby obtaining a biomaterial for skin; the cross-linking agent includes butylene glycol diglycidyl ether, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

22. The method as described in claim 21, characterized in that, The method includes: first cross-linking a collagen-like protein and sodium hyaluronate with butylene glycol diglycidyl ether to form a first network, and then cross-linking with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network, thereby obtaining a biomaterial for skin use; or, first cross-linking a collagen-like protein and sodium hyaluronate with butylene glycol diglycidyl ether to form a first network, and then cross-linking with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to form a second network, thereby obtaining a biomaterial for skin use.

23. The method as described in claim 21 or 22, characterized in that, The method includes the following steps: First dissolution step: Collagen, collagen-like substances, lyophilized collagen powder and / or lyophilized collagen-like substances are dissolved in an alkaline solution and stirred for the first time to obtain a solution; sodium hyaluronate is dissolved in the solution and stirred for the second time to obtain a mixture; butanediol diglycidyl ether is dissolved in the mixture and stirred for the third time to obtain the first cross-linking system; First crosslinking step: The first crosslinking system is allowed to stand for the first time to allow the crosslinking reaction to occur, and the first crosslinking product is obtained; First freeze-drying step: The first cross-linking product is pre-frozen and then freeze-dried for the first time to obtain a sponge-like freeze-dried product; Second dissolution step: Dissolve the spongy lyophilized product in a solvent containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second crosslinking system; or, dissolve the spongy lyophilized product in a solvent containing 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride to obtain a second crosslinking system; Second crosslinking step: The second crosslinking system is allowed to stand for a second time to allow the crosslinking reaction to occur, resulting in the second crosslinking product; Second freeze-drying step: After washing the second cross-linking product, a second freeze-drying is performed to obtain a biomaterial for skin use.

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