Preparation method of an active injection gel capable of tolerating irradiation sterilization

Abstract

The present invention discloses a preparation method of an active injectable gel that can withstand irradiation sterilization. The active injectable gel is composed of a collagen gel extracted from an animal source, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acids compounded in a certain proportion, and serves as a filling material that plays an exogenous filling and endogenous collagen regeneration role. Through the protection of glycerol and amino acids and the method of cryopackaging with liquid nitrogen or dry ice, the collagen gel can still maintain its activity and gel properties after being sterilized by Co60 irradiation, and achieve the effects of sterility and virus inactivation. By adding acellular dermal matrix microparticles and bacterial cellulose, the injection force is increased and the action time of the pure collagen gel is effectively extended to achieve a long-term effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical injection materials, and particularly relates to a preparation method of an active injection gel capable of tolerating irradiation sterilization. Background Art

[0002] Collagen is a nutrient that the human body must supplement to delay aging. As people age, collagen will gradually be lost. Women start to age and lose collagen at the age of 20, and the content decreases year by year. At the age of 25, it enters the peak period of loss. At the age of 40, the content is less than half of that at the age of 18. The grooves and wrinkles on the faces of the elderly are exactly due to the loss of collagen and moisture, resulting in skin collapse. The loss of collagen causes the collagen peptide bonds and elastic networks that support the skin to break, and its helical network structure is immediately damaged. The skin tissue is oxidized, atrophied, and collapsed, and the skin will show aging phenomena such as dryness, wrinkles, and lack of elasticity. Therefore, to delay aging, it is necessary to supplement collagen.

[0003] Medical collagen injection gel is the most effective and convenient way to solve problems such as collapse, wrinkles, and lack of elasticity. At present, there are many collagen injection gel products, but since these products will denature during irradiation sterilization, they are currently commercialized using aseptic processes, which have high requirements for the environment, equipment, and personnel, causing certain limitations and high-cost investments in the production of such products.

[0004] Co60 is the most commonly used terminal sterilization method for medical devices, but it often breaks the chemical bonds or intermolecular forces of some polymer materials, resulting in the denaturation or deterioration of the materials. As a biological polymer, collagen needs to improve its preservation and sterilization techniques to avoid changes in its performance after terminal sterilization. Currently, bovine collagen, porcine collagen, and recombinant collagen have all been industrialized in the field of medical aesthetic injection filling, but their aseptic requirements can only be achieved through the aseptic production process and cannot be achieved through terminal irradiation sterilization. Irradiation sterilization can not only sterilize the terminal products, but also inactivate viruses for xenogeneic collagen, which can improve the safety of collagen implants. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an active injection gel capable of tolerating irradiation sterilization.

[0006] A preparation method of an active injection gel capable of tolerating irradiation sterilization is carried out according to the following steps:

[0007] (1)Select fresh animal skins, remove the surface fat and minced meat, soak in purified water to remove blood, wash, cut into pieces, pulverize in a tissue pulverizer, wash the pulverized tissue with deionized water, soak in 75% ethanol for 18 - 30 h, wash with deionized water, then place the pulverized tissue in deionized water, add pepsin with 0.005 - 0.015 N hydrochloric acid, enzymatically hydrolyze at 23 - 27 °C for 40 - 60 h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 1 - 3 M NaOH, salting - out with saturated NaCl for 8 - 12 h, centrifuge, dialyze the precipitate against deionized water to obtain collagen gel;

[0008] (2)Select fresh animal skins, remove the epidermis, dermal papilla layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.5 - 1.2 mm, place it in a trypsin solution, shake at 35 - 39 °C for 1 - 3 h, wash with purified water 3 - 5 times, 20 min each time;

[0009] (3)Place the matrix treated in step (2) in a 0.5 - 1.5 M NaOH solution, shake at 23 - 27 °C for 0.5 - 1.5 h, wash with purified water 2 - 4 times, 10 min each time;

[0010] (4)Place the matrix treated in step (3) in a 0.05 - 0.15% SDS solution, shake at 23 - 27 °C for 0.5 - 1.5 h, ultrasonically wash with 0.005 - 0.015 M PBS solution 2 - 4 times, 10 min each time;

[0011] (5)Place the matrix treated in step (4) in a TritonX - 100 solution, shake at 23 - 27 °C for 3 - 5 h, ultrasonically wash with 0.005 - 0.015 M PBS solution 4 - 6 times, 10 min each time, and then wash with purified water 2 - 4 times, 20 min each time;

[0012] (6)Put the matrix treated in step (5) into a storage tube, grind it in a grinder, take it out and open the storage tube after grinding to obtain acellular dermal matrix microparticles;

[0013] (7)Mix the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acids evenly, remove air bubbles under reduced pressure and then pour into a syringe;

[0014] (8)Place the syringe in an incubator with a sandwich layer, put dry ice or liquid nitrogen in the sandwich layer of the incubator, close the incubator after placing, perform Co60 irradiation, irradiation dose 15 - 35 KGy, irradiation time 6 - 10 h.

[0015] The animal is one or several of human, non - human primates, fish, birds, cattle, pigs, horses, sheep, dogs, mules, chickens, ducks, geese.

[0016] The addition amount of pepsin described in step (1) is 1-5% of the mass of the animal skin.

[0017] The skin taking in step (2) is carried out by using a DQ-2A 400×900 electric skinning machine.

[0018] The mass concentration of the trypsin solution described in step (2) is 0.1-0.5%.

[0019] The concentration of the TritonX-100 solution described in step (5) is 0.5-2%.

[0020] The grinder described in step (6) is a SPEX 6670 type cryogenic grinder, and the set parameters are: pre-cooling for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min.

[0021] The amino acid is one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0022] The dosage mass ratio of the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acid is 100:(5-10):(5-10):(1-5):(0.5-2).

[0023] The beneficial effects of the present invention: Through the protection of glycerol and amino acids, and by the method of low-temperature encapsulation with liquid nitrogen or dry ice, the collagen gel can still maintain its activity and gel properties after Co60 irradiation sterilization, and achieve the effects of sterility and virus inactivation. By adding acellular dermal matrix microparticles and bacterial cellulose, the injection force is improved and the action time of the pure collagen gel is effectively prolonged, achieving the purpose of long-term effect. Brief Description of the Drawings

[0024] Figure 1 It is a diagram for measuring the thermal denaturation temperature of the gel before irradiation.

[0025] Figure 2 It is a diagram for measuring the thermal denaturation temperature of the gel after irradiation.

[0026] Figure 3 It is a comparison of the gel injection conditions before and after irradiation.

[0027] Figure 4 It is the HE staining result.

[0028] Figure 5 It is the Masson trichrome staining result. Detailed Embodiments

[0029] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive. Example 1

[0030] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0031] (1) Select fresh cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, crush it in a tissue grinder, wash the crushed tissue with deionized water, soak it in 75% ethanol for 24 h, wash it with deionized water, then place the crushed tissue in deionized water, add pepsin with 0.01N hydrochloric acid, the addition amount of pepsin is 3% of the mass of the cowhide, enzymatically hydrolyze at 25 °C for 48 h, centrifuge with a centrifuge to collect the supernatant, adjust it to neutral with 2M NaOH, salting-out with saturated NaCl for 10 h, centrifuge, dialyze the precipitate against deionized water to obtain a collagen gel;

[0032] (2) Select fresh cowhide, use a DQ-2A 400×900 electric skinning machine to take the skin, remove the epidermis, dermal papillary layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.8 mm is placed in a 0.125% trypsin solution, and after shaking treatment at 37 °C for 2 h, wash it 4 times with purified water, 20 min each time;

[0033] (3) Place the matrix treated in step (2) in a 1M NaOH solution, shake it at 25 °C for 1 h, then wash it 3 times with purified water, 10 min each time;

[0034] (4) Place the matrix treated in step (3) in a 0.1% SDS solution, shake it at 25 °C for 1 h, then ultrasonically wash it 3 times with 0.01M PBS solution, 10 min each time;

[0035] (5) Place the matrix treated in step (4) in a 1% TritonX-100 solution, shake it at 25 °C for 4 h, then ultrasonically wash it 5 times with 0.01M PBS solution, 10 min each time, and then wash it 3 times with purified water, 20 min each time;

[0036] (6) Put the matrix treated in step (5) into a storage tube and grind it in a grinder, the grinder is a SPEX6670 type cryogenic grinder, set the parameters: pre-cool for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; after grinding, take it out and open the storage tube to obtain acellular dermal matrix microparticles;

[0037] (7) Mix the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and glycine evenly, degas under reduced pressure, and then pour the mixture into a syringe. The mass ratio of the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acid is 100:8:8:2:1;

[0038] (8) Place the syringe in an incubator with a sandwich layer. Put dry ice or liquid nitrogen in the sandwich layer of the incubator. After placing, close the incubator and perform Co60 irradiation with an irradiation dose of 25 KGy and an irradiation time of 8 h. Example 2

[0039] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0040] (1) Select fresh cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash it, cut it into pieces, pulverize it in a tissue pulverizer, wash the pulverized tissue with deionized water, soak it in 75% ethanol for 20 h, wash it with deionized water, then place the pulverized tissue in deionized water, add pepsin with 0.005 N hydrochloric acid, and the addition amount of pepsin is 1% of the mass of the cowhide. After enzymatic hydrolysis at 23 °C for 60 h, centrifuge to collect the supernatant, adjust it to neutral with 1 M NaOH, salting out with saturated NaCl for 8 h, centrifuge, and dialyze the precipitate against deionized water to obtain a collagen gel;

[0041] (2) Select fresh cowhide, use a DQ-2A 400×900 electric skinning machine to take the skin, remove the epidermis, dermal papilla layer, and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.5 mm. Place it in a 0.25% trypsin solution, shake it at 35 °C for 3 h, then wash it 5 times with purified water, 20 min each time;

[0042] (3) Place the matrix treated in step (2) in a 0.5 M NaOH solution, shake it at 23 °C for 1.5 h, then wash it 4 times with purified water, 10 min each time;

[0043] (4) Place the matrix treated in step (3) in a 0.1% SDS solution, shake it at 23 °C for 1.5 h, then ultrasonically wash it 4 times with a 0.005 M PBS solution, 10 min each time;

[0044] (5) Place the matrix treated in step (4) in a 2% TritonX-100 solution, shake it at 23 °C for 5 h, then ultrasonically wash it 6 times with a 0.005 M PBS solution, 10 min each time, and then wash it 4 times with purified water, 20 min each time;

[0045] (6) Place the matrix processed in step (5) into a storage tube, and grind it in a grinder, where the grinder is a SPEX6670 cryogenic grinder. Set the parameters: pre-cool for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min. After grinding, take out and open the storage tube to obtain acellular dermal matrix microparticles.

[0046] (7) Mix collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and valine evenly, and pour them into a syringe after degassing under reduced pressure. The mass ratio of the amounts of collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acid is 100:5:5:1:0.5.

[0047] (8) Place the syringe in an incubator with a sandwich layer. Put dry ice or liquid nitrogen in the sandwich layer of the incubator. After placing, close the incubator and perform Co60 irradiation with an irradiation dose of 15 KGy and an irradiation time of 10 h. Example 3

[0048] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0049] (1) Select fresh cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, crush it in a tissue grinder, wash the crushed tissue with deionized water, soak it in 75% ethanol for 30 h, wash it with deionized water, then place the crushed tissue in deionized water, add pepsin with 0.015N hydrochloric acid, and the addition amount of pepsin is 5% of the mass of the cowhide. After enzymatic hydrolysis at 27 °C for 40 h, centrifuge to collect the supernatant, adjust it to neutral with 3M NaOH, salting-out with saturated NaCl for 8 h, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel.

[0050] (2) Select fresh cowhide, use a DQ-2A 400×900 electric skinning machine to take the skin, remove the epidermis, dermal papilla layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 1.2 mm. Place it in a 0.35% trypsin solution and shake it at 39 °C for 1 h, then wash it 3 times with purified water for 20 min each time.

[0051] (3) Place the matrix processed in step (2) in a 1.5M NaOH solution and shake it at 27 °C for 0.5 h, then wash it 2 times with purified water for 10 min each time.

[0052] (4) Place the matrix processed in step (3) in a 0.15% SDS solution and shake it at 27 °C for 0.5 h, then ultrasonically wash it 2 times with 0.015M PBS solution for 10 min each time.

[0053] (5) Place the matrix processed in step (4) into a 0.5% Triton X-100 solution, shake it at 27 °C for 3 h, then ultrasonically wash it 4 times with a 0.015 M PBS solution for 10 min each time, and then wash it 2 times with purified water for 20 min each time;

[0054] (6) Put the matrix processed in step (5) into a storage tube and grind it in a grinder. The grinder is a SPEX6670 type cryogenic grinder. Set the parameters: pre-cool for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; After grinding, take it out and open the storage tube to obtain acellular dermal matrix microparticles;

[0055] (7) Mix the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, tyrosine, and cysteine evenly, and pour it into a syringe after degassing under reduced pressure; The mass ratio of the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acids is 100:10:10:5:1:1;

[0056] (8) Place the syringe in an incubator with a sandwich layer. Put dry ice or liquid nitrogen in the sandwich layer of the incubator. After placing it, close the incubator and perform Co60 irradiation with an irradiation dose of 35 KGy and an irradiation time of 6 h. Example 4

[0057] A preparation method of an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0058] (1) Select fresh sheepskin, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, crush it in a tissue grinder, wash the crushed tissue with deionized water, and soak it in 75% ethanol for 24 h, wash it with deionized water, then place the crushed tissue in deionized water, add pepsin with 0.01 N hydrochloric acid, and the addition amount of pepsin is 2.5% of the mass of the sheepskin. After enzymatic hydrolysis at 25 °C for 48 h, centrifuge to collect the supernatant, adjust it to neutral with 2 M NaOH, salting out with saturated NaCl for 10 h, centrifuge, and dialyze the precipitate against deionized water to obtain a collagen gel;

[0059] (2) Select fresh sheepskin, use a DQ-2A 400×900 electric skinning machine to take the skin, remove the epidermis, dermal papilla layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.8 mm. Place it in a 0.25% trypsin solution, shake it at 37 °C for 2 h, then wash it 4 times with purified water for 20 min each time;

[0060] (3) Place the matrix processed in step (2) in a 1 M NaOH solution, shake it at 25 °C for 1 h, then wash it 3 times with purified water for 10 min each time;

[0061] (4) Place the substrate processed in step (3) into a 0.1% SDS solution, shake it at 25 °C for 1 h, then ultrasonically wash it three times with a 0.01 M PBS solution for 10 min each time;

[0062] (5) Place the substrate processed in step (4) into a 1% Triton X-100 solution, shake it at 25 °C for 4 h, then ultrasonically wash it five times with a 0.01 M PBS solution for 10 min each time, and then wash it three times with purified water for 20 min each time;

[0063] (6) Put the substrate processed in step (5) into a storage tube and grind it in a grinder, the grinder is a SPEX6670 type cryogenic grinder, set the parameters: pre-cool for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; after grinding, take it out and open the storage tube to obtain acellular dermal matrix microparticles;

[0064] (7) Mix the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and glycine evenly, and then pour them into a syringe after decompression and defoaming; the mass ratio of the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acids is 100:8:8:2:1;

[0065] (8) Place the syringe in an incubator with a sandwich layer, put dry ice or liquid nitrogen in the sandwich layer of the incubator, close the incubator after placing, and perform Co60 irradiation with an irradiation dose of 25 kGy and an irradiation time of 8 h. Example 5

[0066] A method for preparing an active injection gel resistant to irradiation sterilization is carried out according to the following steps:

[0067] (1) Select fresh pigskin, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, crush it in a tissue grinder, wash the crushed tissue with deionized water, soak it in 75% ethanol for 20 h, wash it with deionized water, then place the crushed tissue in deionized water, add pepsin with 0.005 N hydrochloric acid, the addition amount of pepsin is 1.5% of the mass of the pigskin, enzymatically hydrolyze it at 23 °C for 60 h, centrifuge to collect the supernatant, adjust it to neutral with 1 M NaOH, salting-out with saturated NaCl for 8 h, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0068] (2) Select fresh pigskin, use a DQ-2A 400×900 electric skin cutter to take the skin, remove the epidermis, dermal papilla layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.5 mm, place it in a 0.32% trypsin solution, shake it at 35 °C for 3 h, then wash it five times with purified water for 20 min each time;

[0069] (3) The matrix processed in step (2) is placed in a 0.5 M NaOH solution and shaken at 23 °C for 1.5 h, then washed 4 times with purified water, 10 min each time;

[0070] (4) The matrix processed in step (3) is placed in a 0.1% SDS solution and shaken at 23 °C for 1.5 h, then ultrasonically washed 4 times with a 0.005 M PBS solution, 10 min each time;

[0071] (5) The matrix processed in step (4) is placed in a 2.5% TritonX-100 solution and shaken at 23 °C for 5 h, then ultrasonically washed 6 times with a 0.005 M PBS solution, 10 min each time, and then washed 4 times with purified water, 20 min each time;

[0072] (6) The matrix processed in step (5) is put into a storage tube and ground in a grinder, and the grinder is a SPEX6670 type cryogenic grinder. The set parameters are: pre-cooling for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; After grinding, take out and open the storage tube to obtain acellular dermal matrix microparticles;

[0073] (7) Collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and valine are mixed evenly, and after degassing under reduced pressure, they are poured into a syringe; The dosage mass ratio of the collagen gel, acellular dermal matrix microparticles, bacterial cellulose, glycerol, and amino acid is 100:5:5:1:0.5;

[0074] (8) The syringe is placed in an incubator with a sandwich layer. Dry ice or liquid nitrogen is placed in the sandwich layer of the incubator. After placing, close the incubator and perform Co60 irradiation. The irradiation dose is 15 KGy and the irradiation time is 10 h.

[0075] Comparative Example 1

[0076] A method for preparing an active injection gel resistant to irradiation sterilization is carried out according to the following steps:

[0077] (1) Select fresh cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, crush it in a tissue grinder, wash the crushed tissue with deionized water, soak it in 75% ethanol for 24 h, wash it with deionized water, then place the crushed tissue in deionized water, add pepsin with 0.01 N hydrochloric acid, and the addition amount of pepsin is 3% of the mass of the cowhide. After enzymatic hydrolysis at 25 °C for 48 h, centrifuge to collect the supernatant, adjust it to neutral with 2 M NaOH, salting out with saturated NaCl for 10 h, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0078] (2)Select fresh cowhide, use a DQ-2A 400×900 electric skinning machine to remove the epidermis, dermal papilla layer and subcutaneous fat, leaving the middle dermal reticular layer with a thickness of 0.8 mm. Place it in a 0.125% trypsin solution, shake it at 37°C for 2 h, then wash it 4 times with purified water, 20 min each time;

[0079] (3)Place the substrate treated in step (2) in a 1M NaOH solution, shake it at 25°C for 1 h, then wash it 3 times with purified water, 10 min each time;

[0080] (4)Place the substrate treated in step (3) in a 0.1% SDS solution, shake it at 25°C for 1 h, then ultrasonically wash it 3 times with a 0.01M PBS solution, 10 min each time;

[0081] (5)Place the substrate treated in step (4) in a 1% TritonX-100 solution, shake it at 25°C for 4 h, then ultrasonically wash it 5 times with a 0.01M PBS solution, 10 min each time, and then wash it 3 times with purified water, 20 min each time;

[0082] (6)Put the substrate treated in step (5) into a storage tube and grind it in a grinder. The grinder is a SPEX6670 type cryogenic grinder. Set the parameters: pre-cool for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; After grinding, take it out and open the storage tube to obtain acellular dermal matrix microparticles;

[0083] (7)Mix the collagen gel, acellular dermal matrix microparticles, glycerol, and glycine evenly, and pour them into a syringe after decompression and defoaming; The dosage mass ratio of the collagen gel, acellular dermal matrix microparticles, glycerol, and amino acids is 100:16:2:1;

[0084] (8)Place the syringe in an incubator with a sandwich. Put dry ice or liquid nitrogen in the sandwich of the incubator. After placing it, close the incubator and perform Co60 irradiation with an irradiation dose of 25 KGy and an irradiation time of 8 h.

[0085] Comparative Example 2

[0086] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0087] (1)Select fresh cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash it, cut it into pieces, pulverize it in a tissue pulverizer, wash the pulverized tissue with deionized water, soak it in 75% ethanol for 24 h, wash it with deionized water, then place the pulverized tissue in deionized water, add pepsin with 0.01N hydrochloric acid, and the addition amount of pepsin is 3% of the mass of cowhide. After enzymolysis at 25 °C for 48 h, centrifuge to collect the supernatant, adjust it to neutral with 2M NaOH, salting out with saturated NaCl for 10 h, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0088] (2)Select fresh cowhide, use a DQ-2A 400×900 electric leather cutter to cut the leather, remove the epidermis, dermal papilla layer and subcutaneous fat, and the remaining middle dermal reticular layer with a thickness of 0.8 mm. Place it in a 0.125% trypsin solution, and after shaking treatment at 37 °C for 2 h, wash it 4 times with purified water, 20 min each time;

[0089] (3)Place the matrix treated in step (2) in a 1M NaOH solution, shake it at 25 °C for 1 h, then wash it 3 times with purified water, 10 min each time;

[0090] (4)Place the matrix treated in step (3) in a 0.1% SDS solution, shake it at 25 °C for 1 h, then ultrasonically wash it 3 times with 0.01M PBS solution, 10 min each time;

[0091] (5)Place the matrix treated in step (4) in a 1% TritonX-100 solution, shake it at 25 °C for 4 h, then ultrasonically wash it 5 times with 0.01M PBS solution, 10 min each time, and then wash it 3 times with purified water, 20 min each time;

[0092] (6)Put the matrix treated in step (5) into a storage tube and grind it in a grinder. The grinder is a SPEX6670 type cryogenic grinder, and the set parameters are: pre-cooling for 10 min, the number of cycles is 3, the running time is 2 min, and the cooling time is 2 min; After grinding, take it out and open the storage tube to obtain acellular dermal matrix microparticles;

[0093] (7)Mix collagen gel, bacterial cellulose, glycerol, and glycine evenly, remove bubbles under reduced pressure and then pour it into a syringe; The dosage mass ratio of collagen gel, bacterial cellulose, glycerol, and amino acid is 100:16:2:1;

[0094] (8)Place the syringe in a thermostatic box with a sandwich layer. Put dry ice or liquid nitrogen in the sandwich layer of the thermostatic box. After placing it, close the thermostatic box and perform Co60 irradiation with an irradiation dose of 25 KGy and an irradiation time of 8 h.

[0095] Experimental example:

[0096] (1)DSC measurement: The material was tested using a DSC-60 differential thermal analyzer with argon as the carrier. The measurement temperature range was -40 - 20°C, and the heating rate was 10°C·min -1 , and the object of measurement was the injection gel prepared in step (7) of Example 1; after adding liquid nitrogen or dry ice for cryogenic treatment to the injection gel, irradiation was carried out with an irradiation dose of 25 KGy.

[0097] It can be seen from the DSC test results ( Figure 1-2 ), the thermal denaturation temperature of the gel before irradiation was 63.21°C, and the thermal denaturation temperature of the gel after irradiation was 67.14°C. The temperature difference < 4°C, within the instrument error range, it can be basically judged that the gel did not denature before and after irradiation.

[0098] (2)Injection performance: The injection force was measured using INSTRON4204 at room temperature with a displacement rate of 5 mm / min. The termination condition was L = 200 N, and the load size at uniform injection was recorded. The ideal injection force should be less than 50 N. Each group of experiments was measured in parallel 5 times and the average value was taken. SPSS 24.0 software was used for statistical analysis. The results of measurement data were expressed as x̅±s (mean ± standard deviation). The Kolmogorov-Smirnov test method was used for data normality test. For data that conform to normal distribution, the t-test was used to compare the mean differences between two groups, and P < 0.05 was considered statistically significant.

[0099] The measurement results are shown in Table 1:

[0100] Table 1

[0101]

[0102] (3)Comparison of gel injection conditions: The injection gel prepared in step (7) of Example 1 was placed in 10 1-mL syringes with a 27G needle. Randomly select 5 syringes and put them into an insulated box with a sandwich layer. Dry ice was placed in the sandwich layer for irradiation with an irradiation dose of 25 KGy. After irradiation, the injection experiments before and after irradiation were carried out respectively, and the gel state during the injection process was observed to see if the colloidal injection was not smooth and intermittent due to gel irradiation denaturation.

[0103] The results are as Figure 3 shown. Before and after irradiation, the injection gel could maintain a uniform, smooth and continuous colloid during the injection process without interruption, and the gel did not denature.

[0104] (4)In vivo experiment: To observe the biocompatibility of the gel and its ability to induce tissue regeneration, a subcutaneous implantation test was conducted. Using SD rats as the experimental model, they were anesthetized with 0.3% pentobarbital, and 1 mL of the injection gel prepared in Example 1 was subcutaneously implanted as the experimental group. Hyaluronic acid was used as the control group. Samples were taken on the 28th day. The tissue containing the implant was excised and then fixed in a fixative as a pathological specimen for HE and Masson staining and analysis.

[0105] On the 28th day, the results of HE staining showed that ( Figure 4 ), in the control group, the skin tissue structure was severely abnormal, and a large number of inflammatory cells were visible in the granulation tissue. In the experimental group, a small number of inflammatory cells were visible in the dermis layer, and there were more fibroblasts and new blood vessels compared to the control group. The results of Masson trichrome staining showed that ( Figure 5 ), the collagen density in the experimental group was greater than that in the control group, indicating that after the implantation of the experimental group material, the ability to stimulate and promote collagen regeneration was stronger.

[0106] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for preparing an active injection gel that can withstand irradiation sterilization, characterized in that: Follow these steps: (1) Select fresh animal skin, remove surface fat and minced meat, soak in pure water to remove blood, wash, cut into pieces, and grind in a tissue grinder. Wash the crushed tissue with deionized water, soak in 75% ethanol for 18-30 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.005-0.015N hydrochloric acid, and hydrolyze at 23-27℃ for 40-60 hours. Collect the supernatant by centrifugation, adjust to neutrality with 1-3M NaOH, and salt out with saturated NaCl for 8-12 hours. Centrifuge, dialyze the precipitate with deionized water to obtain collagen gel; (2) Select fresh animal skin, remove the epidermis, dermal papillary layer and subcutaneous fat, and leave the middle dermal reticular layer with a thickness of 0.5-1.2 mm. Place it in a trypsin solution, shake it at 35-39°C for 1-3 hours, and then wash it with purified water 3-5 times, 20 minutes each time; (3) The substrate treated in step (2) is placed in a 0.5-1.5 M NaOH solution at 23-27° C. and shaken for 0.5-1.5 h, and then washed 2-4 times with purified water, 10 min each time; (4) placing the substrate treated in step (3) in a 0.05-0.15% SDS solution, shaking at 23-27°C for 0.5-1.5 h, and then ultrasonically washing with a 0.005-0.015 M PBS solution for 2-4 times, 10 min / time; (5) placing the matrix treated in step (4) in a TritonX-100 solution, shaking at 23-27°C for 3-5 hours, and then ultrasonically washing with a 0.005-0.015M PBS solution for 4-6 times, 10 minutes each time, and then washing with purified water for 2-4 times, 20 minutes each time; (6) placing the matrix treated in step (5) into a storage tube, placing it in a grinder for grinding, taking it out after grinding and opening the storage tube to obtain acellular dermal matrix particles; (7) Mix the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids evenly, decompress and degas, and then pour into the syringe; (8) Place the syringe in an insulated box with a layer of interlayer, and put dry ice or liquid nitrogen in the interlayer of the insulated box. After placement, close the insulated box and perform Co60 irradiation. The irradiation dose is 15-35KGy and the irradiation time is 6-10h.

2. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The animal is one or more of humans, non-human primates, fish, cattle, pigs, horses, sheep, dogs, mules, chickens, ducks, and geese.

3. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The amount of pepsin added in step (1) is 1-5% of the weight of the animal skin.

4. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The skin removal in step (2) is performed using a DQ-2A 400×900 electric skin removal machine.

5. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The mass concentration of the trypsin solution in step (2) is 0.1-0.5%.

6. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The concentration of the TritonX-100 solution in step (5) is 0.5-2%.

7. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The grinder in step (6) is a SPEX 6670 cryo-grinder, and the setting parameters are: precooling for 10 min, number of cycles for 3, running time for 2 min, and cooling time for 2 min.

8. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The amino acid is one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

9. The method for preparing the active injection gel that can withstand radiation sterilization according to claim 1, characterized in that: The mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol and amino acids is 100: (5-10): (5-10): (1-5): (0.5-2).

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