Biological material for skin and preparation method therefor

Abstract

The present application relates to a biological material for skin and a preparation method therefor and pertains to the technical field of biology. The present application provides a biological material for skin. The biological material comprises a collagen analog and sodium hyaluronate. The collagen analog and sodium hyaluronate are first cross-linked by means of BDDE to form a first network and then cross-linked by means of EDC and NHS to form a second network, or the collagen analog and sodium hyaluronate are first cross-linked by means of BDDE to form a first network and then cross-linked by means of DMTMM to form a second network. The biological material uses a non-animal-derived PEGylated collagen analog in place of collagen. Collagen has the effects of promoting tissue and cell growth and promoting cell adhesion; moreover, collagen can maintain a certain level of mechanical performance. The PEGylated collagen analog, while having all the advantages of collagen, is essentially free of animal-derived viruses and immunogenicity.

Description

A biomaterial for skin and a preparation method thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202410543689.6, filed on April 30, 2024, and entitled "A biomaterial for skin and a preparation method thereof", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to a biomaterial for skin and a preparation method thereof, and belongs to the field of biotechnology. BACKGROUND

[0004] Skin is divided into three parts: epidermis, dermis and subcutaneous tissue. Artificial dermis is used to replace the dermis layer of the skin, which is generally divided into two layers: the upper layer is a medical silicone rubber membrane with semi-permeable membrane properties, which plays a role similar to the epidermis, can control water evaporation and prevent microbial invasion; the lower layer is a sponge-like dermal scaffold layer constructed by collagen-chondroitin sulfate, which plays a role as a cell growth scaffold, is conducive to the invasion and growth of vascular endothelial cells and fibroblasts at the transplantation site, forming a scaffold-new capillary-cell complex, which is fully vascularized after 2-3 weeks, and then autologous split-thickness skin can be transplanted, and subsequently, the dermal scaffold will gradually degrade and be replaced by new dermal tissue.

[0005] The main indications of artificial dermis are full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, surgical wounds (donor / graft, post-Mohs surgery, laser surgery, foot disease and wound dehiscence), traumatic wounds (abrasions, lacerations, second-degree burns and skin avulsion wounds), and draining wounds. Artificial dermis can effectively guide the regeneration of new dermis, reduce and inhibit scar proliferation, thereby restoring the elasticity and flexibility of the wound surface and improving the appearance and function. Even in some wound repair, double-layer artificial dermis can directly cover exposed bones and tendons, replacing traditional flap transplantation surgery. The use of artificial dermis on large-area skin defect wounds can early close the wound surface, reduce the loss of wound body fluids including water and plasma proteins, thereby reducing the consumption of body nutrients. For wounds after tumor resection, it can observe whether the tumor recurs earlier, reducing the risk of tumor residue. At the same time, after vascularization of artificial dermis, only split-thickness skin needs to be transplanted, not only the survival rate of skin grafts is high, but also the donor site heals quickly with less damage and lighter scar formation. Compared with flap transplantation surgery, it is time-saving and has low risk of anesthesia.

[0006] At present, the main representative of the commercially available artificial dermis products is Integra's double-layer artificial dermis and Anika's Hyalomatrix, which are mainly composed of biological materials, and Polynovo's NovoSorb BTM, which is mainly composed of high molecular chemical materials. Among them, the main component of Integra's double-layer artificial dermis is bovine collagen type I and shark 6-sulfate chondroitin. After mixing and precipitating the two components, the collagen is crosslinked with glutaraldehyde, freeze-dried to form a scaffold, and then a layer of silica gel is compounded, and then stored in phosphate buffer at 2-30°C. The materials come from animals, which have certain animal-derived virus risk, especially the collagen used is derived from cattle, which needs to be imported from New Zealand. The raw material processing process is complex, the source is limited, and it will constitute an immunogenic risk. At the same time, the crosslinking agent used is glutaraldehyde, which has high toxicity and safety problems. The main component of Anika's Hyalomatrix is hyaluronic acid derivative benzyl hyaluronate. The benzyl hyaluronate is formed into a dermal layer by electrospinning. The material belongs to a derivative of biological materials, and has low similarity to the main components of human dermis (the main components of human dermis are collagen, elastin, reticular protein, hyaluronic acid, vascular hair follicles and sweat glands sebaceous glands), and has poor biocompatibility. The main component of Polynovo's NovoSorb BTM is degradable polyurethane, which has lower similarity to the main components of human dermis and has poorer biocompatibility. Therefore, it is urgent to find an artificial dermis that can eliminate the risk of animal-derived viruses, immunogenicity, high safety, and consistency with the components of human dermis as much as possible, and has better biocompatibility to overcome the defects of existing artificial dermis products. SUMMARY

[0007] To solve the above problems, the present application provides a biological material for skin, which comprises collagen and / or collagen-like protein; the collagen and / or collagen-like protein is crosslinked by a crosslinking agent to form a network; the crosslinking agent comprises butanediol diglycidyl ether (BDDE), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (DMTMM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS).

[0008] In an embodiment of the present application, the biological material for skin further comprises sodium hyaluronate.

[0009] In an embodiment of the present application, the biomaterial for skin comprises collagen-like protein and sodium hyaluronate; the collagen-like protein and sodium hyaluronate are first crosslinked by butanediol diglycidyl ether to form a first network, and then crosslinked by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network, or the collagen-like protein and sodium hyaluronate are first crosslinked by butanediol diglycidyl ether to form a first network, and then crosslinked by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to form a second network.

[0010] In an embodiment of the present application, the method for preparing the biomaterial for skin comprises the following steps:

[0011] The first dissolving step: collagen, collagen-like protein, collagen lyophilized powder and / or collagen-like protein lyophilized powder are dissolved in an alkaline solution for the first stirring to obtain a dissolving solution; sodium hyaluronate is dissolved in the dissolving solution for the second stirring to obtain a mixed solution; butanediol diglycidyl ether is dissolved in the mixed solution for the third stirring to obtain a first crosslinking system;

[0012] The first crosslinking step: the first crosslinking system is subjected to the first standing to occur crosslinking reaction to obtain a first crosslinking product;

[0013] The first lyophilization step: the first crosslinking product is pre-frozen and then subjected to the first lyophilization to obtain a sponge-like lyophilized product;

[0014] The second dissolving step: the sponge-like lyophilized product is dissolved in a solvent added with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second crosslinking system; or the sponge-like lyophilized product is dissolved in a solvent added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to obtain a second crosslinking system;

[0015] The second crosslinking step: the second crosslinking system is subjected to the second standing to occur crosslinking reaction to obtain a second crosslinking product;

[0016] The second lyophilization step: the second crosslinking product is washed and then subjected to the second lyophilization to obtain the biomaterial for skin.

[0017] In an embodiment of the present application, the collagen-like protein is pegylated collagen-like protein; and the collagen-like protein lyophilized powder is pegylated collagen-like protein lyophilized powder.

[0018] In an embodiment of the present application, in the first dissolving step, the concentration of collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL.

[0019] In one embodiment of the present application, the concentration of the collagen or collagen-like protein in the dissolving solution in the first dissolving step is 100-120 g / mL.

[0020] In one embodiment of the present application, the concentration of the sodium hyaluronate in the mixing solution in the first dissolving step is 100-300 mg / mL.

[0021] In one embodiment of the present application, the concentration of the sodium hyaluronate in the mixing solution in the first dissolving step is 100-120 g / mL.

[0022] In one embodiment of the present application, the molar ratio of the butanediol diglycidyl ether to the sodium hyaluronate in the first cross-linking system in the first dissolving step is 2:1-10:1.

[0023] In one embodiment of the present application, the molar ratio of the butanediol diglycidyl ether to the sodium hyaluronate in the first cross-linking system in the first dissolving step is 2:1-6:1.

[0024] In one embodiment of the present application, the first stirring in the first dissolving step is performed for 0.5-10 h at 20-65 °C and at a rotation speed of 100-2000 r / min.

[0025] In one embodiment of the present application, the first stirring in the first dissolving step is performed for 1-5 h at 35-45 °C and at a rotation speed of 400-600 r / min.

[0026] In one embodiment of the present application, the second stirring in the first dissolving step is performed for 0.5-10 h at 20-45 °C and at a rotation speed of 100-2000 r / min.

[0027] In one embodiment of the present application, the second stirring in the first dissolving step is performed for 1.5-10 h at 30-45 °C and at a rotation speed of 400-600 r / min.

[0028] In one embodiment of the present application, the third stirring in the first dissolving step is performed for 0.5-10 h at 20-65 °C and at a rotation speed of 100-2000 r / min.

[0029] In one embodiment of the present application, the third stirring in the first dissolving step is performed for 1.5-3 h at 30-45 °C and at a rotation speed of 400-1000 r / min.

[0030] In one embodiment of the present application, the pH of the alkaline solution is 10-14.

[0031] In one embodiment of the present application, the pH of the alkaline solution is 12-14.

[0032] In one embodiment of the present application, the alkaline solution comprises a sodium hydroxide solution, a potassium hydroxide solution, a sodium bicarbonate solution and / or a sodium carbonate solution.

[0033] In one embodiment of the present application, the alkaline solution is a sodium hydroxide solution with a pH of 10-14.

[0034] In one embodiment of the present application, the alkaline solution is a sodium hydroxide solution with a pH of 12-14.

[0035] In one embodiment of the present application, in the first cross-linking step, the first standing time is 0.5-10 h and the temperature is 20-55°C.

[0036] In one embodiment of the present application, in the first cross-linking step, the first standing time is 3-4 h and the temperature is 30-40°C.

[0037] In one embodiment of the present application, in the first freeze-drying step, the pre-freezing time is 4-24 h and the temperature is -10 to -40°C.

[0038] In one embodiment of the present application, in the first freeze-drying step, the pre-freezing time is 10-14 h and the temperature is -25 to -30°C.

[0039] In one embodiment of the present application, in the first freeze-drying step, the first freeze-drying time is 12-24 h and the temperature is -15 to -40°C.

[0040] In one embodiment of the present application, in the first freeze-drying step, the first freeze-drying time is 14-18 h and the temperature is -20 to -40°C.

[0041] In one embodiment of the present application, in the second dissolving 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.

[0042] In one embodiment of the present application, in the second dissolving 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.

[0043] In an embodiment of the present application, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 10-100 mmol / L.

[0044] In an embodiment of the present application, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 20-100 mmol / L.

[0045] In an embodiment of the present application, the solvent comprises water and / or an organic solvent; the organic solvent comprises ethanol, isopropanol, n-butanol and / or pentanediol.

[0046] In an embodiment of the present application, the solvent of 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 of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride is water.

[0047] In an embodiment of the present application, in the second cross-linking step, the second standing time is 12-48 h and the temperature is 20-40℃.

[0048] In an embodiment of the present application, in the second cross-linking step, the second standing time is 24-48 h and the temperature is 20-30℃.

[0049] In an embodiment of the present application, in the second freeze-drying step, the washing time is 1-48 h.

[0050] In an embodiment of the present application, in the second freeze-drying step, the washing time is 4-24 h.

[0051] In an embodiment of the present application, in the second freeze-drying step, the washing comprises washing with water for injection.

[0052] In an embodiment of the present application, in the second freeze-drying step, the second freeze-drying time is 12-24 h and the temperature is -15 to -40℃.

[0053] In an embodiment of the present application, in the second freeze-drying step, the second freeze-drying time is 14-18 h and the temperature is -15 to -40℃.

[0054] In an embodiment of the present application, the method for preparing the pegylated collagen-like protein comprises the following steps:

[0055] The reaction step is to react the collagen-like protein and the polyethylene glycol derivative under the conditions of pH 4.0-10.0 and temperature 2-40°C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0056] In an embodiment of the present application, the method for preparing the lyophilized powder of the pegylated collagen-like protein comprises the following steps:

[0057] The reaction step is to react the collagen-like protein and the polyethylene glycol derivative under the conditions of pH 4.0-10.0 and temperature 2-40°C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0058] The lyophilization step is to lyophilize the reaction product containing the pegylated collagen-like protein to obtain the lyophilized powder of the pegylated collagen-like protein.

[0059] In an embodiment of the present application, the reaction step is to react the collagen-like protein and the polyethylene glycol derivative under the conditions of pH 4.0-10.0 and temperature 2-40°C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0060] In an embodiment of the present application, the reaction step is to react the collagen-like protein and the polyethylene glycol derivative under the conditions of pH 6.0-8.0 and temperature 2-8°C for 5-8 h to obtain a reaction product containing the pegylated collagen-like protein.

[0061] In an embodiment of the present application, in the reaction step, the molar ratio of the collagen-like protein to the polyethylene glycol derivative is 1-16:1.

[0062] In an embodiment of the present application, in the reaction step, the molar ratio of the collagen-like protein to the polyethylene glycol derivative is 8-12:1.

[0063] In an embodiment of the present application, in the reaction step, the reaction solvent of the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.

[0064] In an embodiment of the present application, the concentration of the dilute hydrochloric acid solution is 1-10 mmol / L; and the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 by a dilute alkali solution.

[0065] In an embodiment of the present application, the dilute alkali solution is a sodium hydroxide solution or ammonia water with pH 9.0-11.0.

[0066] In an embodiment of the present application, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg / mL.

[0067] In one embodiment of the present application, the concentration of the collagen-like protein in the reaction solvent in the reaction step is 8-10 mg / mL.

[0068] In one embodiment of the present application, the freeze-drying step is a step of mixing the reaction product containing the PEGylated collagen-like protein and a freeze-drying protective agent and then freeze-drying to obtain the PEGylated collagen-like protein freeze-dried powder.

[0069] In one embodiment of the present application, the mass ratio of the reaction product containing the PEGylated collagen-like protein and the freeze-drying protective agent in the freeze-drying step is 1:1-10.

[0070] In one embodiment of the present application, the freeze-drying protective agent is one or more of mannitol, sucrose or alanine.

[0071] In one embodiment of the present application, the method further comprises a purification step after the reaction step and before the freeze-drying step; the purification step is a step of filtering and retaining the substances with a molecular weight of 30000 Da or more in the reaction product containing the PEGylated collagen-like protein at a temperature of 2-8°C to obtain the PEGylated collagen-like protein.

[0072] In one embodiment of the present application, the filtration is dialysis or ultrafiltration.

[0073] In one embodiment of the present application, 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), i.e.

[0074] The amino acid sequence of SEQ ID NO. 1 is:

[0075] 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-Hyp-Gly-OH

[0076] In one embodiment of the present application, the PEG derivative comprises PEG-40k and PEG-20k.

[0077] In one embodiment of the present application, the PEG derivative consists of PEG-40k and PEG-20k.

[0078] In one embodiment of the present application, the molar ratio of PEG-40k to PEG-20k is 0.5-6:1.

[0079] In one embodiment of the present application, the molar ratio of PEG-40k to PEG-20k is 2-3:1.

[0080] In one embodiment of the present application, the number of activated groups of PEG-40k is one or more of eight-arm, four-arm, two-arm or one-arm; the number of activated groups of PEG-20k is one or more of eight-arm, four-arm, two-arm or one-arm.

[0081] In one embodiment of the present application, the number of activated groups of PEG-40k is four-arm or eight-arm; the number of activated groups of PEG-20k is four-arm or eight-arm.

[0082] In one embodiment of the present application, the activated group of PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; the activated group of PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.

[0083] In one embodiment of the present application, the activated group of PEG-40k is -MAL; the activated group of PEG-20k is -MAL.

[0084] In one embodiment of the present application, one end of the collagen-like protein is connected with a connecting peptide; in the reaction step, the polyethylene glycol derivative is modified on the connecting peptide through the activated group to obtain a reaction product containing the pegylated collagen-like protein.

[0085] In one embodiment of the present application, the connecting peptide is connected to the N-terminus of the collagen-like protein.

[0086] In one embodiment of the present application, the modification site of the polyethylene glycol derivative on the connecting peptide is one or more of thiol, amino, carboxyl or imidazole.

[0087] In one embodiment of the present application, the parent nucleus conformation of the pegylated collagen-like protein is one or more of HG or TP.

[0088] In one embodiment of the present application, the parent nucleus conformation of the polyethylene glycol derivative in the pegylated collagen-like protein is TP.

[0089] In one embodiment of the present application, the amino acid configuration of the collagen-like protein in the pegylated collagen-like protein is one or more of D-form or L-form.

[0090] In an embodiment of the present application, the molecular weight of the pegylated collagen-like protein is 15000-75000 Da.

[0091] In an embodiment of the present application, the molecular weight of the pegylated collagen-like protein is 30000-75000 Da.

[0092] In an embodiment of the present application, the biomaterial for skin comprises artificial dermis, artificial epidermis and / or artificial skin.

[0093] In an embodiment of the present application, the artificial skin comprises a combination of dermis and epidermis, a combination of dermis and subcutaneous tissue, and / or a combination of epidermis, dermis and subcutaneous tissue.

[0094] In an embodiment of the present application, the artificial skin is a functional artificial skin constructed using seed cells.

[0095] In an embodiment of the present application, the seed cells comprise immune cells, fibroblasts, vascular endothelial cells, vascular smooth muscle cells and / or hair follicle stem cells; the immune cells comprise lymphocytes and / or phagocytes.

[0096] The present application also provides a method for preparing a biomaterial for skin, comprising: crosslinking collagen and / or collagen-like protein with a crosslinking agent to form a network; the crosslinking agent comprises butanediol diglycidyl ether, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide, to obtain a biomaterial for skin.

[0097] In an embodiment of the present application, the method comprises: first crosslinking collagen-like protein and sodium hyaluronate with butanediol diglycidyl ether to form a first network, and then crosslinking with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a second network, to obtain a biomaterial for skin, or first crosslinking collagen-like protein and sodium hyaluronate with butanediol diglycidyl ether to form a first network, and then crosslinking with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to form a second network, to obtain a biomaterial for skin.

[0098] In an embodiment of the present application, the method comprises the following steps:

[0099] The first dissolving step: dissolving the collagen, the collagen-like protein, the collagen freeze-dried powder and / or the collagen-like protein freeze-dried powder in the alkaline solution to perform the first stirring to obtain a dissolving solution; dissolving the sodium hyaluronate in the dissolving solution to perform the second stirring to obtain a mixed solution; dissolving the butanediol diglycidyl ether in the mixed solution to perform the third stirring to obtain a first cross-linking system;

[0100] The first cross-linking step: performing the first standing of the first cross-linking system to occur the cross-linking reaction to obtain a first cross-linking product;

[0101] The first freeze-drying step: performing the first freeze-drying of the first cross-linking product after pre-freezing to obtain a sponge freeze-dried product;

[0102] The second dissolving step: dissolving the sponge freeze-dried product in the solvent added with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second cross-linking system; or dissolving the sponge freeze-dried product in the solvent added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to obtain a second cross-linking system;

[0103] The second cross-linking step: performing the second standing of the second cross-linking system to occur the cross-linking reaction to obtain a second cross-linking product;

[0104] The second freeze-drying step: performing the second freeze-drying of the second cross-linking product after cleaning to obtain the biomaterial for skin.

[0105] In an embodiment of the present application, the collagen-like protein is a pegylated collagen-like protein; and the collagen-like protein freeze-dried powder is a pegylated collagen-like protein freeze-dried powder.

[0106] In an embodiment of the present application, in the first dissolving step, the concentration of the collagen or the collagen-like protein in the dissolving solution is 100-300 mg / mL.

[0107] In an embodiment of the present application, in the first dissolving step, the concentration of the collagen or the collagen-like protein in the dissolving solution is 100-120 g / mL.

[0108] In an embodiment of the present application, in the first dissolving step, the concentration of the sodium hyaluronate in the mixed solution is 100-300 mg / mL.

[0109] In an embodiment of the present application, in the first dissolving step, the concentration of the sodium hyaluronate in the mixed solution is 100-120 g / mL.

[0110] In one embodiment of the present application, in the first dissolving step, the molar ratio of butanediol diglycidyl ether to sodium hyaluronate in the first crosslinking system is 2:1 to 10:1.

[0111] In one embodiment of the present application, in the first dissolving step, the molar ratio of butanediol diglycidyl ether to sodium hyaluronate in the first crosslinking system is 2:1 to 6:1.

[0112] In one embodiment of the present application, in the first dissolving step, the first stirring is performed for 0.5 to 10 hours at 20 to 65°C at a rotation speed of 100 to 2000 r / min.

[0113] In one embodiment of the present application, in the first dissolving step, the first stirring is performed for 1 to 5 hours at 35 to 45°C at a rotation speed of 400 to 600 r / min.

[0114] In one embodiment of the present application, in the first dissolving step, the second stirring is performed for 0.5 to 10 hours at 20 to 45°C at a rotation speed of 100 to 2000 r / min.

[0115] In one embodiment of the present application, in the first dissolving step, the second stirring is performed for 1.5 to 10 hours at 30 to 45°C at a rotation speed of 400 to 600 r / min.

[0116] In one embodiment of the present application, in the first dissolving step, the third stirring is performed for 0.5 to 10 hours at 20 to 65°C at a rotation speed of 100 to 2000 r / min.

[0117] In one embodiment of the present application, in the first dissolving step, the third stirring is performed for 1.5 to 3 hours at 30 to 45°C at a rotation speed of 400 to 1000 r / min.

[0118] In one embodiment of the present application, the pH of the alkaline solution is 10 to 14.

[0119] In one embodiment of the present application, the pH of the alkaline solution is 12 to 14.

[0120] In one embodiment of the present application, the alkaline solution comprises a sodium hydroxide solution, a potassium hydroxide solution, a sodium bicarbonate solution, and / or a sodium carbonate solution.

[0121] In one embodiment of the present application, the alkaline solution is a sodium hydroxide solution having a pH of 10 to 14.

[0122] In one embodiment of the present application, the alkaline solution is a sodium hydroxide solution with a pH of 12-14.

[0123] In one embodiment of the present application, in the first cross-linking step, the first standing time is 0.5-10 h and the temperature is 20-55°C.

[0124] In one embodiment of the present application, in the first cross-linking step, the first standing time is 3-4 h and the temperature is 30-40°C.

[0125] In one embodiment of the present application, in the first freeze-drying step, the pre-freezing time is 4-24 h and the temperature is -10 to -40°C.

[0126] In one embodiment of the present application, in the first freeze-drying step, the pre-freezing time is 10-14 h and the temperature is -25 to -30°C.

[0127] In one embodiment of the present application, in the first freeze-drying step, the first freeze-drying time is 12-24 h and the temperature is -15 to -40°C.

[0128] In one embodiment of the present application, in the first freeze-drying step, the first freeze-drying time is 14-18 h and the temperature is -20 to -40°C.

[0129] In one embodiment of the present application, in the second dissolving 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.

[0130] In one embodiment of the present application, in the second dissolving 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.

[0131] In one embodiment of the present application, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 10-100 mmol / L.

[0132] In one embodiment of the present application, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 20-100 mmol / L.

[0133] In an embodiment of the present application, the solvent comprises water and / or an organic solvent; the organic solvent comprises ethanol, isopropanol, n-butanol and / or pentanediol.

[0134] In an embodiment of the present application, the solvent for the 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 the 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride is water.

[0135] In an embodiment of the present application, in the second cross-linking step, the second standing time is 12-48 h and the temperature is 20-40 °C.

[0136] In an embodiment of the present application, in the second cross-linking step, the second standing time is 24-48 h and the temperature is 20-30 °C.

[0137] In an embodiment of the present application, in the second freeze-drying step, the washing time is 1-48 h.

[0138] In an embodiment of the present application, in the second freeze-drying step, the washing time is 4-24 h.

[0139] In an embodiment of the present application, in the second freeze-drying step, the washing comprises washing with water for injection.

[0140] In an embodiment of the present application, in the second freeze-drying step, the second freeze-drying time is 12-24 h and the temperature is -15 to -40 °C.

[0141] In an embodiment of the present application, in the second freeze-drying step, the second freeze-drying time is 14-18 h and the temperature is -15 to -40 °C.

[0142] In an embodiment of the present application, the method for preparing the pegylated collagen-like protein comprises the following steps:

[0143] The reaction step: the collagen-like protein and the polyethylene glycol derivative are reacted at a pH of 4.0-10.0 and a temperature of 2-40 °C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0144] In an embodiment of the present application, the method for preparing the pegylated collagen-like protein freeze-dried powder comprises the following steps:

[0145] The reaction step is that the collagen-like protein and the polyethylene glycol derivative are reacted at pH 4.0-10.0 and temperature 2-40 °C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0146] The freeze-drying step is that the reaction product containing the pegylated collagen-like protein is freeze-dried to obtain the pegylated collagen-like protein freeze-dried powder.

[0147] In an embodiment of the present application, the reaction step is that the collagen-like protein and the polyethylene glycol derivative are reacted at pH 4.0-10.0 and temperature 2-40 °C for 1-48 h to obtain a reaction product containing the pegylated collagen-like protein.

[0148] In an embodiment of the present application, the reaction step is that the collagen-like protein and the polyethylene glycol derivative are reacted at pH 6.0-8.0 and temperature 2-8 °C for 5-8 h to obtain a reaction product containing the pegylated collagen-like protein.

[0149] In an embodiment of the present application, in the reaction step, the molar ratio of the collagen-like protein to the polyethylene glycol derivative is 1-16:1.

[0150] In an embodiment of the present application, in the reaction step, the molar ratio of the collagen-like protein to the polyethylene glycol derivative is 8-12:1.

[0151] In an embodiment of the present application, in the reaction step, the reaction solvent of the collagen-like protein and the polyethylene glycol derivative is water or dilute hydrochloric acid solution.

[0152] In an embodiment of the present application, the concentration of the dilute hydrochloric acid solution is 1-10 mmol / L; and the pH of the dilute hydrochloric acid solution is adjusted to 6.0-8.0 by a dilute alkali solution.

[0153] In an embodiment of the present application, the dilute alkali solution is a sodium hydroxide solution or ammonia water with pH 9.0-11.0.

[0154] In an embodiment of the present application, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 1-15 mg / mL.

[0155] In an embodiment of the present application, in the reaction step, the feeding concentration of the collagen-like protein in the reaction solvent is 8-10 mg / mL.

[0156] In an embodiment of the present application, the freeze-drying step is that the reaction product containing the pegylated collagen-like protein is mixed with a freeze-drying protective agent and then freeze-dried to obtain the pegylated collagen-like protein freeze-dried powder.

[0157] In one embodiment of the present application, the mixture of the reaction product containing the PEGylated collagen-like protein and the freeze-drying protective agent in the freeze-drying step has a mass ratio of 1:1 to 10.

[0158] In one embodiment of the present application, the freeze-drying protective agent is one or more of mannitol, sucrose or alanine.

[0159] In one embodiment of the present application, the method further comprises a purification step after the reaction step and before the freeze-drying step; the purification step is: filtering to retain substances with a molecular weight of 30000 Da or more in the reaction product containing the PEGylated collagen-like protein at a temperature of 2 to 8°C to obtain the PEGylated collagen-like protein.

[0160] In one embodiment of the present application, the filtering is dialysis or ultrafiltration.

[0161] In one embodiment of the present application, 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), i.e.

[0162] The amino acid sequence of SEQ ID NO. 1 is:

[0163] 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-Hyp-Gly-OH

[0164] In one embodiment of the present application, the PEG derivative comprises PEG-40k and PEG-20k.

[0165] In one embodiment of the present application, the PEG derivative consists of PEG-40k and PEG-20k.

[0166] In one embodiment of the present application, the molar ratio of PEG-40k to PEG-20k is 0.5 to 6:1.

[0167] In one embodiment of the present application, the molar ratio of PEG-40k to PEG-20k is 2 to 3:1.

[0168] In one embodiment of the present application, the number of activated groups of the PEG-40k is one or more of eight-arm, four-arm, two-arm or one-arm; and the number of activated groups of the PEG-20k is one or more of eight-arm, four-arm, two-arm or one-arm.

[0169] In one embodiment of the present application, the number of activated groups of the PEG-40k is four-arm or eight-arm; and the number of activated groups of the PEG-20k is four-arm or eight-arm.

[0170] In one embodiment of the present application, the activated group of the PEG-40k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC; and the activated group of the PEG-20k is one or more of -MAL, -NHS, -SG, -SPA, -SS or -EDC.

[0171] In one embodiment of the present application, the activated group of the PEG-40k is -MAL; and the activated group of the PEG-20k is -MAL.

[0172] In one embodiment of the present application, one end of the collagen-like protein is connected with a connecting peptide; and in the reaction step, the polyethylene glycol derivative is modified on the connecting peptide through the activated group to obtain a reaction product containing the polyethylene glycolated collagen-like protein.

[0173] In one embodiment of the present application, the connecting peptide is connected to the N-terminus of the collagen-like protein.

[0174] In one embodiment of the present application, the modification site of the polyethylene glycol derivative on the connecting peptide is one or more of thiol, amino, carboxyl or imidazole.

[0175] In one embodiment of the present application, the parent nucleus conformation of the polyethylene glycolated collagen-like protein is one or more of HG or TP.

[0176] In one embodiment of the present application, the parent nucleus conformation of the polyethylene glycol derivative in the polyethylene glycolated collagen-like protein is TP.

[0177] In one embodiment of the present application, the amino acid configuration of the collagen-like protein in the polyethylene glycolated collagen-like protein is one or more of D-form or L-form.

[0178] In one embodiment of the present application, the molecular weight of the polyethylene glycolated collagen-like protein is 15000-75000 Da.

[0179] In one embodiment of the present application, the molecular weight of the polyethylene glycolated collagen-like protein is 30000-75000 Da.

[0180] In one embodiment of the present application, the biomaterial for skin comprises artificial dermis, artificial epidermis and / or artificial skin.

[0181] In one embodiment of the present application, the artificial skin comprises a combination of dermis and epidermis, a combination of dermis and subcutaneous tissue, and / or a combination of epidermis, dermis and subcutaneous tissue.

[0182] In one embodiment of the present application, the artificial skin is a functional artificial skin constructed using seed cells.

[0183] In one embodiment of the present application, the seed cells comprise immune cells, fibroblasts, vascular endothelial cells, vascular smooth muscle cells and / or hair follicle stem cells; the immune cells comprise lymphocytes and / or phagocytes.

[0184] The present application also provides a method for promoting the growth of skin cells and immune cells, comprising applying the biomaterial to the skin.

[0185] In one embodiment of the present application, the skin cells are fibroblasts and vascular endothelial cells, and the immune cells are lymphocytes.

[0186] The present application also provides a method for promoting the growth and repair of skin tissue, comprising applying the biomaterial to the skin.

[0187] The present application also provides the use of the biomaterial in promoting the growth and repair of skin tissue. The technical solution of the present application has the following advantages:

[0188] The present application provides a biomaterial for skin, which comprises collagen-like protein and sodium hyaluronate; the collagen-like protein and sodium hyaluronate are first cross-linked by butanediol diglycidyl ether to form a first network, and then cross-linked by 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 by butanediol diglycidyl ether to form a first network, and then cross-linked by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to form a second network. The biomaterial for skin has the following advantages:

[0189] First, non-animal source pegylated collagen-like protein is used instead of collagen, which has the effects of promoting tissue and cell growth, promoting cell adhesion, and maintaining certain mechanical properties. The pegylated collagen-like protein fundamentally eliminates animal-derived viruses and immunogenicity on the basis of all the advantages of collagen.

[0190] Second, the addition of sodium hyaluronate, which is contained in the skin itself, in the pegylated collagen makes the composition of the combination more consistent with the components of human dermis, and has better biocompatibility. Moreover, hyaluronic acid participates in every step of wound repair and has a non-specific effect on cell receptors (CD44 / RHAMM and ICAM-1), which can effectively promote tissue growth and repair.

[0191] Third, the cross-linking agents (butanediol diglycidyl ether, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide) used are non-cytotoxic and have high safety.

[0192] Fourth, the pore size is suitable for cell growth, which can promote the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes,

[0193] Therefore, the biomaterial for skin can be used as artificial dermis, artificial epidermis or artificial skin, and has wide application prospects in the field of skin regeneration and tissue repair.

[0194] Further, the preparation method of the biomaterial for skin comprises a first dissolving step, a first cross-linking step, a first freeze-drying step, a second dissolving step, a second cross-linking step and a second freeze-drying step. In the first dissolving step, collagen, collagen-like protein, collagen freeze-dried powder and / or collagen-like protein freeze-dried powder are dissolved in an alkaline solution for the first time to obtain a dissolving solution. Sodium hyaluronate is dissolved in the dissolving solution for the second time to obtain a mixed solution. Butanediol diglycidyl ether is dissolved in the mixed solution for the third time to obtain a first cross-linking system. In the first cross-linking step, the first cross-linking system is allowed to stand for the first time to generate a cross-linking reaction to obtain a first cross-linking product. In the 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. In the second dissolving step, the sponge-like freeze-dried product is dissolved in a solvent added with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second cross-linking system, or the sponge-like freeze-dried product is dissolved in a solvent added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to obtain a second cross-linking system. In the second cross-linking step, the second cross-linking system is allowed to stand for the second time to generate a cross-linking reaction to obtain a second cross-linking product. In the second freeze-drying step, the second cross-linking product is washed and then freeze-dried for the second time to obtain the biomaterial for skin. The biomaterial for skin prepared by the preparation method promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0195] Further, the collagen-like protein is a pegylated collagen-like protein; and the collagen-like protein lyophilized powder is a pegylated collagen-like protein lyophilized powder. The biological material for skin prepared under this setting has better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0196] Further, in the first dissolving step, the concentration of the collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL. The biological material for skin can be successfully prepared under this setting.

[0197] Further, in the first dissolving step, the concentration of the collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL. The biological material for skin can be successfully prepared under this setting.

[0198] Further, in the first dissolving step, the concentration of the collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL. The biological material for skin can be successfully prepared under this setting.

[0199] Further, in the first dissolving step, the concentration of the collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL. The biological material for skin can be successfully prepared under this setting.

[0200] Further, in the first dissolving step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first cross-linking system is 2:1-10:1. The biological material for skin can be successfully prepared under this setting.

[0201] Further, in the first dissolving step, the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first cross-linking system is 2:1-10:1. The biological material for skin can be successfully prepared under this setting.

[0202] Further, in the first dissolving step, the time of the first stirring is 0.5-10 h, the temperature is 20-65℃, and the rotation speed is 100-2000 r / min. The biological material for skin can be successfully prepared under this setting.

[0203] Further, in the first dissolving step, the first stirring is performed for 1-5 hours at 40-45°C and at a rotation speed of 400-600 r / min. The biological material for skin prepared under this condition has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for the growth and repair of skin tissue.

[0204] Further, in the first dissolving step, the second stirring is performed for 0.5-10 hours at 20-45°C and at a rotation speed of 100-2000 r / min. The biological material for skin can be successfully prepared under this condition.

[0205] Further, in the first dissolving step, the second stirring is performed for 1.5-10 hours at 30-45°C and at a rotation speed of 400-600 r / min. The biological material for skin prepared under this condition has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for the growth and repair of skin tissue.

[0206] Further, in the first dissolving step, the third stirring is performed for 0.5-10 hours at 20-65°C and at a rotation speed of 100-2000 r / min. The biological material for skin can be successfully prepared under this condition.

[0207] Further, in the first dissolving step, the third stirring is performed for 1.5-3 hours at 30-45°C and at a rotation speed of 400-1000 r / min. The biological material for skin prepared under this condition has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for the growth and repair of skin tissue.

[0208] Further, the pH of the alkaline solution is 10-14. The biological material for skin can be successfully prepared under this condition.

[0209] Further, the pH of the alkaline solution is 12-14. The biological material for skin prepared under this condition has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for the growth and repair of skin tissue.

[0210] Further, in the first cross-linking step, the first standing is performed for 0.5-10 hours at 20-55°C. The biological material for skin can be successfully prepared under this condition.

[0211] Further, in the first cross-linking step, the first standing time is 3-4 h and the temperature is 30-40℃. Under this setting, the biomaterial for skin prepared promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0212] Further, in the first freeze-drying step, the pre-freezing time is 4-24 h and the temperature is -10--40℃. Under this setting, the biomaterial for skin can be successfully prepared.

[0213] Further, in the first freeze-drying step, the pre-freezing time is 10-14 h and the temperature is -25--30℃. Under this setting, the biomaterial for skin prepared promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0214] Further, in the first freeze-drying step, the pre-freezing time is 10-14 h and the temperature is -25--30℃. Under this setting, the biomaterial for skin prepared promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0215] Further, in the first freeze-drying step, the pre-freezing time is 10-14 h and the temperature is -25--30℃. Under this setting, the biomaterial for skin prepared promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0216] Further, in the second dissolving 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, the biomaterial for skin can be successfully prepared.

[0217] Further, in the second dissolving 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 biomaterial for skin prepared promotes the growth of skin cells such as fibroblasts and vascular endothelial cells, and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0218] Further, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 10-100 mmol / L. Under this setting, the biomaterial for skin can be successfully prepared.

[0219] Further, in the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride in the solvent is 20-100 mmol / L. The biomaterial for skin prepared under this setting has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0220] Further, in the second cross-linking step, the time for the second standing is 12-48 h and the temperature is 20-40℃. The biomaterial for skin can be successfully prepared under this setting.

[0221] Further, in the second cross-linking step, the time for the second standing is 24-48 h and the temperature is 20-30℃. The biomaterial for skin prepared under this setting has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0222] Further, in the second freeze-drying step, the time for the washing is 1-48 h. The biomaterial for skin can be successfully prepared under this setting.

[0223] Further, in the second freeze-drying step, the time for the washing is 4-24 h. The biomaterial for skin prepared under this setting has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair.

[0224] Further, in the second freeze-drying step, the time for the second freeze-drying is 12-24 h and the temperature is -15 to -40℃. The biomaterial for skin can be successfully prepared under this setting.

[0225] Further, in the second freeze-drying step, the time for the second freeze-drying is 14-18 h and the temperature is -15 to -40℃. The biomaterial for skin prepared under this setting has a better effect of promoting the growth of skin cells such as fibroblasts and vascular endothelial cells and immune cells such as lymphocytes, and is helpful for skin tissue growth and repair. BRIEF DESCRIPTION OF DRAWINGS

[0226] Figure 1: Environmental scanning electron microscope image of artificial dermis 1.

[0227] Figure 2: Cell survival rate after culture of different cells with artificial dermis. DETAILED DESCRIPTION

[0228] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the content and protection scope of the application, and do not constitute a limitation on the content and protection scope of the application, and any person who obtains any product identical or similar to the application under the enlightenment of the application or combines the application with other prior art features falls within the protection scope of the application.

[0229] The specific experimental steps or conditions are not specified in the following examples, and can be performed according to the conventional experimental steps or conditions described in the literature in the art. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained on the market. In the following examples, the synthesis of the collagen-like protein and the connection between the collagen-like protein and the connecting peptide are completed by Shanghai AmbioPharm.InC. The PEG derivatives in the following examples are purchased from Xiamen Senobio Biotech Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (DMTMM) and butanediol diglycidyl ether (BDDE) in the following examples are purchased from SIGMA; sodium hyaluronate in the following examples is purchased from Huaxi Biotechnology Co., Ltd.; pre-freezing and freeze-drying in the following examples are completed by FD-1A-50 freeze-dryer purchased from Shanghai Jinpu Electronics Technology Co., Ltd.; stirring in the following examples is completed by a magnetic stirrer purchased from Shanghai Titan Science and Technology Co., Ltd.

[0230] Example 1: An artificial dermis and a preparation method thereof

[0231] The present embodiment provides an artificial dermis, and a preparation method thereof, which comprises the following steps:

[0232] The first dissolution step: the polyethylene glycol collagen-like protein freeze-dried powder (the preparation of the polyethylene glycol collagen-like protein freeze-dried powder is specifically described in the embodiment 1 of the patent application with the publication number CN115990969A) is dissolved in a sodium hydroxide solution (the solvent is water) with a pH of 12, so that the concentration of the polyethylene glycol collagen-like protein in the sodium hydroxide solution is 100 mg / mL, and then the solution is stirred at a temperature of 40℃ and a speed of 400 r / min for 2 hours for the first time to obtain a dissolution solution; the sodium hyaluronate is dissolved in the dissolution solution, so that the concentration of the sodium hyaluronate in the dissolution solution is 100 mg / mL, and then the solution is stirred at a temperature of 40℃ and a speed of 400 r / min for 2 hours for the second time to obtain a mixed solution; the butanediol diglycidyl ether is dissolved in the mixed solution, so that the molar ratio of the butanediol diglycidyl ether to the sodium hyaluronate in the mixed solution is 4:1, and then the solution is stirred at a temperature of 40℃ and a speed of 400 r / min for 2 hours for the third time to obtain a first cross-linking system;

[0233] First cross-linking step: the first cross-linking system was first left to stand at a temperature of 40℃ for 3h to cause cross-linking reaction, to obtain a first cross-linking product;

[0234] First freeze-drying step: the first cross-linking product was poured into a freeze-drying tray, pre-frozen at a temperature of -25℃ for 12h, and then first freeze-dried at a temperature of -25℃ and a vacuum degree of 75Pa for 16h, to obtain a sponge-like freeze-dried product;

[0235] Second dissolving step: the sponge-like freeze-dried product was dissolved in a 95%(v / v) aqueous ethanol solution to which 60mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60mmol / L N-hydroxysuccinimide were added, so that the sponge-like freeze-dried product was soaked in the 95%(v / v) aqueous ethanol solution, to obtain a second cross-linking system;

[0236] Second cross-linking step: the second cross-linking system was second left to stand at a temperature of 25℃ for 24h to cause cross-linking reaction, to obtain a second cross-linking product;

[0237] Second freeze-drying step: the second cross-linking product was washed in water for injection for 12h, and then second freeze-dried at a temperature of -25℃ and a vacuum degree of 75Pa for 16h, to obtain an artificial dermis (this artificial dermis was named as artificial dermis 1).

[0238] Example 2: An artificial dermis and preparation thereof

[0239] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the concentration (100mg / mL) of the pegylated collagen in the first dissolving step is replaced by 20mg / mL, 40mg / mL, 80mg / mL, 120mg / mL, 200mg / mL, and 300mg / mL, respectively.

[0240] The above artificial dermis is sequentially named as artificial dermis 2-7.

[0241] Example 3: An artificial dermis and preparation thereof

[0242] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the concentration (100mg / mL) of the sodium hyaluronate in the first dissolving step is replaced by 20mg / mL, 40mg / mL, 60mg / mL, 80mg / mL, 120mg / mL, 200mg / mL, and 300mg / mL, respectively.

[0243] The above artificial dermis is sequentially named as artificial dermis 8-14.

[0244] Example 4: An artificial dermis and preparation thereof

[0245] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the molar ratio of butanediol diglycidyl ether and sodium hyaluronate in the first dissolving step (4:1) is replaced by 0.5:1, 1:1, 2:1, 6:1, 8:1, 10:1, respectively.

[0246] The above artificial dermis is sequentially designated as artificial dermis 15 to 20.

[0247] Example 5: An artificial dermis and preparation thereof

[0248] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first stirring time in the first dissolving step (2h) is replaced by 0.5h, 1h, 1.5h, 3h, 5h, 10h, respectively.

[0249] The above artificial dermis is sequentially designated as artificial dermis 21 to 26.

[0250] Example 6: An artificial dermis and preparation thereof

[0251] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first stirring temperature in the first dissolving step (40℃) is replaced by 20℃, 25℃, 30℃, 35℃, 45℃, 55℃, 65℃, respectively.

[0252] The above artificial dermis is sequentially designated as artificial dermis 27 to 33.

[0253] Example 7: An artificial dermis and preparation thereof

[0254] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first stirring speed in the first dissolving step (400r / min) is replaced by 100r / min, 200r / min, 600r / min, 1000r / min, 2000r / min, respectively.

[0255] The above artificial dermis is sequentially designated as artificial dermis 34 to 38.

[0256] Example 8: An artificial dermis and preparation thereof

[0257] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the second stirring time in the first dissolving step (2h) is replaced by 0.5h, 1h, 1.5h, 3h, 5h, 10h, respectively.

[0258] The artificial dermis described above is sequentially designated as artificial dermis 39-44.

[0259] Example 9: An artificial dermis and preparation thereof

[0260] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the pH (12) of the sodium hydroxide solution in the first dissolution step is replaced by 7, 8, 9, 10, 11, 13, and 14, respectively.

[0261] The artificial dermis described above is sequentially designated as artificial dermis 45-51.

[0262] Example 10: An artificial dermis and preparation thereof

[0263] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the second stirring temperature (40℃) in the first dissolution step is replaced by 20℃, 25℃, 30℃, 35℃, 45℃, 55℃, and 65℃, respectively.

[0264] The artificial dermis described above is sequentially designated as artificial dermis 52-58.

[0265] Example 11: An artificial dermis and preparation thereof

[0266] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the second stirring speed (400 r / min) in the first dissolution step is replaced by 100 r / min, 200 r / min, 600 r / min, and 1000 r / min, respectively.

[0267] The artificial dermis described above is sequentially designated as artificial dermis 59-63.

[0268] Example 12: An artificial dermis and preparation thereof

[0269] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and the third stirring time (2 h) in the first dissolution step is replaced by 0.5 h, 1 h, 1.5 h, 3 h, 5 h, and 10 h, respectively.

[0270] The artificial dermis described above is sequentially designated as artificial dermis 64-69.

[0271] Example 13: An artificial dermis and preparation thereof

[0272] The present example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the third stirring temperature in the first dissolution step (40°C) is replaced by 20°C, 25°C, 30°C, 35°C, 45°C, 55°C, and 65°C, respectively.

[0273] The above artificial dermis is sequentially designated as artificial dermis 70 to 76.

[0274] Example 14: An artificial dermis and preparation thereof

[0275] The present example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the third stirring speed in the first dissolution step (400 r / min) is replaced by 100 r / min, 200 r / min, 600 r / min, 1000 r / min, and 2000 r / min, respectively.

[0276] The above artificial dermis is sequentially designated as artificial dermis 77 to 81.

[0277] Example 15: An artificial dermis and preparation thereof

[0278] The present example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first standing time in the first cross-linking step (3 h) is replaced by 0.5 h, 1 h, 2 h, 4 h, 5 h, and 10 h, respectively.

[0279] The above artificial dermis is sequentially designated as artificial dermis 82 to 87.

[0280] Example 16: An artificial dermis and preparation thereof

[0281] The present example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first standing temperature in the first cross-linking step (40°C) is replaced by 25°C, 30°C, 35°C, 45°C, and 55°C, respectively.

[0282] The above artificial dermis is sequentially designated as artificial dermis 88 to 92.

[0283] Example 17: An artificial dermis and preparation thereof

[0284] The present example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the second standing time in the second cross-linking step (24 h) is replaced by 6 h, 8 h, 12 h, 36 h, and 48 h, respectively.

[0285] The above artificial dermis is sequentially designated as artificial dermis 93 to 97.

[0286] Example 18: An artificial dermis and preparation thereof

[0287] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the second standing temperature (25℃) in the second cross-linking step is replaced by 4℃, 20℃, 30℃, 35℃, 40℃, respectively.

[0288] The above artificial dermis is sequentially designated as artificial dermis 98-102.

[0289] Example 19: An artificial dermis and preparation thereof

[0290] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the pre-freezing time (12h) in the first freeze-drying step is replaced by 4h, 8h, 18h, 24h, respectively.

[0291] The above artificial dermis is sequentially designated as artificial dermis 103-106.

[0292] Example 20: An artificial dermis and preparation thereof

[0293] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the pre-freezing temperature (-25℃) in the first freeze-drying step is replaced by -10℃, -15℃, -20℃, -30℃, -35℃, -40℃, respectively.

[0294] The above artificial dermis is sequentially designated as artificial dermis 107-112.

[0295] Example 21: An artificial dermis and preparation thereof

[0296] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first freeze-drying time (16h) in the first freeze-drying step is replaced by 6h, 8h, 10h, 14h, 18h, 24h, respectively.

[0297] The above artificial dermis is sequentially designated as artificial dermis 113-118.

[0298] Example 22: An artificial dermis and preparation thereof

[0299] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the first freeze-drying temperature (-25℃) in the first freeze-drying step is replaced by -10℃, -15℃, -20℃, -30℃, -35℃, -40℃, respectively.

[0300] The above artificial dermis is sequentially designated as artificial dermis 119-124.

[0301] Example 23: An artificial dermis and preparation thereof

[0302] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the second freeze-drying time (16 h) in the second freeze-drying step is replaced by 6 h, 8 h, 10 h, 14 h, 18 h, and 24 h, respectively.

[0303] The above artificial dermis is sequentially designated as artificial dermis 125 to 130.

[0304] Example 24: An artificial dermis and preparation thereof

[0305] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the second freeze-drying temperature (-25℃) in the second freeze-drying step is replaced by -10℃, -15℃, -20℃, -30℃, -35℃, and -40℃, respectively.

[0306] The above artificial dermis is sequentially designated as artificial dermis 131 to 136.

[0307] Example 25: An artificial dermis and preparation thereof

[0308] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the washing time (12 h) in the second freeze-drying step is replaced by 4 h, 6 h, 8 h, 14 h, 16 h, and 24 h, respectively.

[0309] The above artificial dermis is sequentially designated as artificial dermis 137 to 142.

[0310] Example 26: An artificial dermis and preparation thereof

[0311] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the 95% (v / v) aqueous ethanol solution to which 60 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mmol / L N-hydroxysuccinimide are added in the second dissolving step is replaced by:

[0312] a 95% (v / v) aqueous ethanol solution to which 10 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mmol / L N-hydroxysuccinimide are added;

[0313] a 95% (v / v) aqueous ethanol solution to which 20 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 20 mmol / L N-hydroxysuccinimide are added;

[0314] 95% (v / v) ethanol aqueous solution added with 40 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 40 mmol / L N-hydroxysuccinimide;

[0315] 95% (v / v) ethanol aqueous solution added with 80 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 80 mmol / L N-hydroxysuccinimide;

[0316] 95% (v / v) ethanol aqueous solution added with 100 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mmol / L N-hydroxysuccinimide.

[0317] The artificial dermis described above is sequentially designated as artificial dermis 143-147.

[0318] Example 27: An artificial dermis and preparation thereof

[0319] This example provides an artificial dermis, which is based on the artificial dermis 1 of Example 1, and in which the 95% (v / v) ethanol aqueous solution added with 60 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mmol / L N-hydroxysuccinimide in the second dissolution step is replaced with:

[0320] a DMTMN aqueous solution having a concentration of 10 mmol / L;

[0321] a DMTMN aqueous solution having a concentration of 20 mmol / L;

[0322] a DMTMN aqueous solution having a concentration of 40 mmol / L;

[0323] a DMTMN aqueous solution having a concentration of 60 mmol / L;

[0324] a DMTMN aqueous solution having a concentration of 80 mmol / L;

[0325] a DMTMN aqueous solution having a concentration of 100 mmol / L.

[0326] The artificial dermis described above is sequentially designated as artificial dermis 148-153.

[0327] Comparative Example 1: An artificial dermis and preparation thereof

[0328] The comparative example provides an artificial dermis, which is an Integra product, according to the literature "I V Yannas, 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.", the collagen and chondroitin sulfate artificial scaffold pore size is 20-125 μm, the seeded autologous dermal cells and epidermal cells have morphological activity, the main material is bovine collagen type I and 6-chondroitin sulfate, and the preparation method is described in US patent US4060081A.

[0329] Experimental Example 1: Effect of preparation process on the performance of artificial dermis

[0330] The experimental example provides an experiment of the effect of the preparation process on the performance of artificial dermis, and the experimental process is as follows:

[0331] Experiment 1: According to 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 collagen-GAG scaffolds, Biomaterials, Volume 25, Issue 6, 2004, Pages 1077-1086, ISSN 0142-9612.", the pore size of artificial dermis 1-68 is measured by environmental scanning electron microscope (QUANTA 200 of Thermo Fisher). In order to ensure the true pore size of the artificial dermis in the wet state, the artificial dermis is cut into 1 cm x 1 cm size before measurement, then the artificial dermis is immersed (i.e. soaked) in PBS buffer (0.00144 g / 100 mL of KH2PO4 and 0.01062 g / 100 mL of Na2HPO4 are added to 0.9 g / 100 mL of sodium chloride solution, and then 0.1 M sodium hydroxide is used to adjust the pH to 7.4 to obtain the PBS buffer) for 20 minutes, and finally the artificial dermis is fixed with conductive glue without dripping water, the electron microscope magnification is 800 times, and the pore size is measured by Image J scale, the measurement results are shown in Table 1 and Figure 1.

[0332] Experiment two: in vitro cell test was conducted on artificial skin 1, 17 and 51 to detect their cytotoxicity, cell proliferation ability and cell adhesion ability, and the specific detection method was as follows:

[0333] The artificial skin was cut into a disc with a diameter of 1 cm, then immersed (i.e. soaked) in PBS buffer for 20 minutes to obtain the sample to be tested; the cells (human fibroblasts and vascular endothelial cells, purchased from Changzhou Huayang Chemical Co., Ltd.) were inoculated into a 24-well plate at a seeding amount of 2.5x10 5 The sample to be tested was added to the 24-well plate; after inoculation, the 24-well plate was placed in a cell incubator at 37°C, 5% (v / v) CO2 for culture; after 1 day (for detecting cytotoxicity) and 5 days (for detecting cell proliferation ability), the 24-well plate was taken out of the cell incubator, the sample to be tested was taken out of the 24-well plate and transferred to a new 24-well plate; after the transfer was completed, MTT solution (purchased from Changzhou Huayang Chemical Co., Ltd.) with a concentration of 1 mg / mL was added to the 24-well plate containing the sample to be tested at an addition amount of 0.5 mL per well; after addition, the 24-well plate was placed in a cell incubator at 37°C, 5% (v / v) CO2 for incubation; after 2 h of incubation, the 24-well plate was taken out of the cell incubator, isopropyl alcohol was added to the 24-well plate at an addition amount of 1 mL per well for dissolution; after addition, the solution in the 24-well plate was first mixed with a shaker, then the 24-well plate was placed into an enzyme label instrument, the absorbance of each well was measured at 570 nm wavelength (subtracting the background of the blank group without cell growth), and the cell survival rate after culture of different cells and artificial skin was calculated according to the measurement results, the measurement results are shown in Tables 2-3, and the calculation results are shown in Figure 2; wherein the calculation formula of cell survival rate is: cell survival rate % = 100% x (OD sample group-OD blank group) / OD blank group.

[0334] As can be seen from Table 1 and Figure 1, the artificial skin scaffold has a pore size in the range of 11-104μm, according to the literature “I V Yannas, 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.” query, the pore size suitable for fibroblast and vascular endothelial cell growth is 20-125μm, and part of the embodiments meet the optimal pore size range, so the cells can proliferate and grow inside the scaffold.

[0335] From Table 2-3 and Figure 2, it can be seen that both vascular endothelial cells and fibroblasts proliferated after being seeded in artificial dermis for 5 days, and the cells in artificial dermis group proliferated more than the control (medium + cells), which indicates that artificial dermis is suitable for cell growth, and cells can proliferate and grow after being immersed in artificial dermis scaffold.

[0336] Table 1 Average pore size of artificial dermis 1-15

[0337] Table 2 Increase in absorbance of vascular endothelial cells seeded in artificial dermis and cultured for different time

[0338] Table 3 Increase in absorbance of human fibroblasts seeded in artificial dermis and cultured for different time

[0339] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A biomaterial for skin, characterized by, The biomaterial for skin includes collagen and / or collagen-like protein; the collagen and / or collagen-like protein is cross-linked by a cross-linking agent to form a network; the cross-linking agent includes butanediol diglycidyl ether, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

2. The biomaterial of claim 1, wherein, The biomaterial for skin further includes sodium hyaluronate.

3. The biomaterial of claim 1 or 2, wherein The biomaterial for skin includes collagen-like protein and sodium hyaluronate; the collagen-like protein and sodium hyaluronate are first cross-linked by butanediol diglycidyl ether to form a first network, and then cross-linked by 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 by butanediol diglycidyl ether to form a first network, and then cross-linked by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to form a second network.

4. The biomaterial according to any one of claims 1 to 3, wherein The preparation method of the biomaterial for skin includes the following steps: A first dissolving step: dissolving collagen, collagen-like protein, collagen lyophilized powder and / or collagen-like protein lyophilized powder in an alkaline solution to perform a first stirring to obtain a dissolving solution; dissolving sodium hyaluronate in the dissolving solution to perform a second stirring to obtain a mixed solution; and dissolving butanediol diglycidyl ether in the mixed solution to perform a third stirring to obtain a first cross-linking system; A first cross-linking step: allowing the first cross-linking system to be first placed to occur a cross-linking reaction to obtain a first cross-linking product; A first lyophilizing step: pre-freezing the first cross-linking product and then performing a first lyophilizing to obtain a sponge-like lyophilized product; A second dissolving step: dissolving the sponge-like lyophilized product in a solvent added with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second cross-linking system, or dissolving the sponge-like lyophilized product in a solvent added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to obtain a second cross-linking system; A second cross-linking step: allowing the second cross-linking system to be second placed to occur a cross-linking reaction to obtain a second cross-linking product; A second lyophilizing step: cleaning the second cross-linking product and then performing a second lyophilizing to obtain the biomaterial for skin.

5. The biomaterial of claim 4, wherein The collagen-like protein is a pegylated collagen-like protein; and the collagen-like protein lyophilized powder is a pegylated collagen-like protein lyophilized powder.

6. The biomaterial of claim 4 or 5, wherein In the first dissolving step, the concentration of collagen or collagen-like protein in the dissolving solution is 100-300 mg / mL.

7. Biomaterial according to any of claims 4 to 6, characterized in that In the first dissolving step, the concentration of sodium hyaluronate in the mixed solution is 100-300 mg / mL.

8. Biomaterial according to any one of claims 4 to 7, characterised in that In the first dissolving step, the molar ratio of butanediol diglycidyl ether to sodium hyaluronate in the first cross-linking system is 2:1-10:

1.

9. Biomaterial according to any one of claims 4 to 8, characterised in that, In the first dissolving step, the first stirring is performed for 0.5-10 h at a temperature of 20-65 ℃ and a rotation speed of 100-2000 r / min.

10. Biomaterial according to any one of claims 4 to 9, characterized in that, The third stirring in the first dissolving step is performed for 0.5-10 hours at 20-65℃ and at a rotation speed of 100-2000 r / min.

11. Biomaterial according to any of claims 4 to 10, characterized in that The third stirring in the first dissolving step is performed for 0.5-10 hours at 20-65℃ and at a rotation speed of 100-2000 r / min.

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

13. Biomaterial according to any of claims 4 to 12, characterized in that The first standing in the first cross-linking step is performed for 0.5-10 hours at 20-55℃.

14. Biomaterial according to any one of claims 4 to 13, characterised in that The pre-freezing in the first freeze-drying step is performed for 4-24 hours at -10--40℃.

15. Biomaterial according to any one of claims 4 to 14, characterized in that The first freeze-drying in the first freeze-drying step is performed for 12-24 hours at -15--40℃.

16. Biomaterial according to any one of claims 4 to 15, characterized in that In the second dissolving 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. Biomaterial according to any of claims 4 to 16, characterized in that In the second dissolving step, the concentration of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in the solvent is 10-100 mmol / L.

18. Biomaterial according to any one of claims 4 to 17, characterized in that The second standing in the second cross-linking step is performed for 12-48 hours at 20-40℃.

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

20. Biomaterial according to any of claims 4 to 19, characterized in that The second freeze-drying in the second freeze-drying step is performed for 12-24 hours at -15--40℃.

21. A method of preparing a biomaterial for use on skin, characterized by, The method comprises: cross-linking collagen and / or collagen-like protein with a cross-linking agent to form a network, thereby obtaining a biomaterial for skin; and the cross-linking agent comprises butanediol diglycidyl ether, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

22. The method of claim 21, wherein, The method comprises: cross-linking collagen and / or collagen-like protein with a cross-linking agent to form a network, thereby obtaining a biomaterial for skin; and the cross-linking agent comprises butanediol diglycidyl ether, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

23. The method of claim 21 or 22, wherein, The method comprises the following steps: The first dissolving step: dissolving collagen, collagen-like protein, collagen freeze-dried powder and / or collagen-like protein freeze-dried powder in an alkaline solution to perform first stirring, thereby obtaining a dissolving solution; dissolving sodium hyaluronate in the dissolving solution to perform second stirring, thereby obtaining a mixed solution; and dissolving butanediol diglycidyl ether in the mixed solution to perform third stirring, thereby obtaining a first cross-linking system; The first cross-linking step: performing first standing on the first cross-linking system to perform cross-linking reaction, thereby obtaining a first cross-linking product; The first freeze-drying step: pre-freezing the first cross-linking product and then performing first freeze-drying, thereby obtaining a sponge-like freeze-dried product; the sponge-like freeze-dried product is dissolved in a solvent added with 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a second cross-linking system; or the sponge-like freeze-dried product is dissolved in a solvent added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to obtain a second cross-linking system; the second cross-linking step: the second cross-linking system is subjected to a second standing to occur a cross-linking reaction to obtain a second cross-linking product; the second freeze-drying step: the second cross-linking product is washed and subjected to a second freeze-drying to obtain a biomaterial for skin.

24. A method of promoting growth of skin cells and immune cells, comprising, The method comprises applying to the skin the biomaterial of any one of claims 1-20 or the biomaterial prepared by the method of any one of claims 21-23.

25. The method of claim 24, wherein, The skin cells are fibroblasts and vascular endothelial cells, and the immune cells are lymphocytes.

26. A method of promoting skin tissue growth and repair, comprising, The method comprises applying to the skin the biomaterial of any one of claims 1-20 or the biomaterial prepared by the method of any one of claims 21-23.

27. Use of the biomaterial of any one of claims 1-20 or the biomaterial prepared by the method of any one of claims 21-23 in promoting growth and repair of skin tissue.

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