Preparation method of active injection gel capable of resisting irradiation sterilization

Abstract

The invention discloses a preparation method of active injection gel capable of resisting irradiation sterilization. The active injection gel is a filling material which is prepared by compounding collagen gel extracted from an animal source, acellular dermal matrix particles, bacterial cellulose, glycerol and amino acid according to a certain proportion and has the effects of exogenous filling and endogenous collagen regeneration. According to the collagen gel disclosed by the invention, the collagen gel can still keep the activity and the gel property after being subjected to Co60 irradiation sterilization by virtue of protection of glycerol and amino acid and a low-temperature packaging manner by virtue of liquid nitrogen or dry ice, and the effects of sterility and virus inactivation are achieved; and the injection force is improved, the action time of the pure collagen gel is effectively prolonged, and the long-acting purpose is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical injection materials, and in particular relates to a method for preparing an active injection gel capable of withstanding radiation sterilization. Background Art

[0002] Collagen is a nutrient that the human body must replenish to delay aging. As we age, collagen will gradually be lost. Women's collagen begins to age and lose 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 wrinkles on the faces of the elderly are caused by the loss of collagen and water, which causes the skin to collapse. The loss of collagen causes the collagen peptide bonds and elastic network that support the skin to break, and its spiral network structure is then destroyed. The skin tissue is oxidized, atrophied, and collapsed, and the skin will show aging phenomena such as dryness, wrinkles, sagging and inelasticity. Therefore, to delay aging, collagen must be supplemented.

[0003] Medical collagen injection gel is the most effective and convenient way to solve problems such as collapse, wrinkles, and inelasticity. There are many collagen injection gel products currently, but because such products will denature during irradiation sterilization, they are currently produced using aseptic processes. The environment, equipment, and personnel requirements are very high, which has caused certain limitations and high cost investment in the production of such products.

[0004] Co60 is the most commonly used terminal sterilization method for medical devices, but it often destroys 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 technology to avoid changes in its performance after terminal sterilization. At present, bovine collagen, porcine collagen, and recombinant collagen have all been industrialized in the field of medical beauty injection filling, but their sterility requirements can only be achieved through aseptic production processes and cannot be achieved through terminal irradiation sterilization. Irradiation sterilization can not only sterilize the terminal product, but also inactivate viruses for heterologous collagen, which can improve the safety of collagen implants. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing an active injection gel which can withstand irradiation sterilization.

[0006] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0007] (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°C 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, and dialyze the precipitate against deionized water to obtain collagen gel;

[0008] (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;

[0009] (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;

[0010] (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;

[0011] (5) placing the substrate treated in step (4) in a TritonX-100 solution, shaking at 23-27° C. for 3-5 h, and then ultrasonically washing it with a 0.005-0.015 M PBS solution for 4-6 times, 10 min / time, and then washing it with purified water for 2-4 times, 20 min / time;

[0012] (6) placing the matrix treated in step (5) into a material storage tube, placing it in a grinder for grinding, and taking it out after grinding and opening the material storage tube to obtain acellular dermal matrix particles;

[0013] (7) Mix the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids evenly, decompress and degas, and then inject into a syringe;

[0014] (8) The syringe is placed in an insulated box with a sandwich layer, and dry ice or liquid nitrogen is placed in the sandwich layer of the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 15-35 KGy and the irradiation time is 6-10 h.

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

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

[0017] The skin removal in step (2) is performed using a DQ-2A 400×900 electric skin removal machine.

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

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

[0020] The grinder in step (6) is a SPEX 6670 cryo-grinder, and the setting parameters are: precooling for 10 min, number of cycles is 3, running time is 2 min, and 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 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).

[0023] The beneficial effects of the present invention are as follows: by protecting with glycerol and amino acids and low-temperature packaging with liquid nitrogen or dry ice, the collagen gel can maintain its activity and gel properties after Co60 irradiation sterilization, and achieve the effects of sterility and virus inactivation. In addition, by adding acellular dermal matrix particles and bacterial cellulose, the injection force is improved and the action time of the pure collagen gel is effectively prolonged, thereby achieving the purpose of long-term effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the thermal denaturation temperature of the gel before irradiation.

[0025] Figure 2 This is a graph showing the thermal denaturation temperature of the irradiated gel.

[0026] Figure 3 Comparison of gel push before and after irradiation.

[0027] Figure 4 HE staining results.

[0028] Figure 5 This is the result of Masson's trichrome staining. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] Example 1

[0031] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0032] (1) Select fresh cowhide, 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 24 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.01N hydrochloric acid, and the amount of pepsin added is 3% of the weight of the cowhide. After enzymatic hydrolysis at 25°C for 48 hours, centrifuge and collect the supernatant, adjust to neutral with 2M NaOH, salt out with saturated NaCl for 10 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0033] (2) Fresh cowhide was selected and the skin was removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.8 mm was placed in a 0.125% trypsin solution and shaken at 37°C for 2 h. The skin was then washed 4 times with purified water, 20 min / time.

[0034] (3) The substrate treated in step (2) was placed in a 1 M NaOH solution, shaken at 25° C. for 1 h, and then washed with purified water three times, 10 min each time;

[0035] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 25° C. for 1 h, and then ultrasonically washing with a 0.01 M PBS solution for 3 times, 10 min each time;

[0036] (5) placing the substrate treated in step (4) in a 1% TritonX-100 solution, shaking at 25°C for 4 h, and then ultrasonically washing it with a 0.01M PBS solution for 5 times, 10 min / time, and then washing it with purified water for 3 times, 20 min / time;

[0037] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0038] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and glycine evenly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids is 100:8:8:2:1;

[0039] (8) The syringe is placed in an insulated box with a layer, and dry ice or liquid nitrogen is placed in the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 25 KGy and the irradiation time is 8 h.

[0040] Example 2

[0041] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0042] (1) Select fresh cowhide, 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 20 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.005N hydrochloric acid, and the amount of pepsin added is 1% of the weight of the cowhide. After enzymatic hydrolysis at 23°C for 60 hours, centrifuge and collect the supernatant, adjust to neutrality with 1M NaOH, salt out with saturated NaCl for 8 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0043] (2) Fresh cowhide was selected and the skin was removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.5 mm was placed in a 0.25% trypsin solution and shaken at 35°C for 3 h. The skin was then washed with purified water for 5 times, 20 min / time.

[0044] (3) The substrate treated in step (2) was placed in a 0.5 M NaOH solution at 23° C. and shaken for 1.5 h, and then washed with purified water 4 times, 10 min each time;

[0045] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 23° C. for 1.5 h, and then ultrasonically washing with a 0.005 M PBS solution for 4 times, 10 min per time;

[0046] (5) placing the substrate treated in step (4) in a 2% TritonX-100 solution, shaking at 23°C for 5 h, and then ultrasonically washing it with a 0.005M PBS solution for 6 times, 10 min / time, and then washing it with purified water for 4 times, 20 min / time;

[0047] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0048] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and valine uniformly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids is 100:5:5:1:0.5;

[0049] (8) The syringe is placed in an insulated box with a sandwich layer, and dry ice or liquid nitrogen is placed in the sandwich layer of the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 15 KGy and the irradiation time is 10 h.

[0050] Example 3

[0051] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0052] (1) Select fresh cowhide, remove fat and minced meat from the surface, 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 30 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.015N hydrochloric acid, and the amount of pepsin added is 5% of the weight of the cowhide. After enzymatic hydrolysis at 27°C for 40 hours, centrifuge and collect the supernatant, adjust to neutrality with 3M NaOH, salt out with saturated NaCl for 8 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0053] (2) Fresh cowhide was selected and the skin was removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 1.2 mm was placed in a 0.35% trypsin solution and shaken at 39°C for 1 h. The skin was then washed three times with purified water, 20 min each time.

[0054] (3) The substrate treated in step (2) was placed in a 1.5 M NaOH solution at 27° C. and shaken for 0.5 h, and then washed twice with purified water, 10 min each time;

[0055] (4) placing the substrate treated in step (3) in a 0.15% SDS solution, shaking at 27° C. for 0.5 h, and then ultrasonically washing the substrate twice with a 0.015 M PBS solution, 10 min each time;

[0056] (5) placing the substrate treated in step (4) in a 0.5% TritonX-100 solution, shaking at 27°C for 3 h, and then ultrasonically washing it with a 0.015M PBS solution for 4 times, 10 min each time, and then washing it with purified water for 2 times, 20 min each time;

[0057] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0058] (7) Mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, tyrosine, and cysteine ​​uniformly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids is 100:10:10:5:1:1;

[0059] (8) The syringe is placed in an insulated box with a sandwich layer, and dry ice or liquid nitrogen is placed in the sandwich layer of the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 35 KGy and the irradiation time is 6 h.

[0060] Example 4

[0061] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0062] (1) Select fresh sheepskin, 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 24 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.01N hydrochloric acid, and the amount of pepsin added is 2.5% of the weight of the sheepskin. After enzymatic hydrolysis at 25°C for 48 hours, centrifuge and collect the supernatant, adjust to neutrality with 2M NaOH, salt out with saturated NaCl for 10 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0063] (2) Fresh sheepskin was selected and removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.8 mm was placed in a 0.25% trypsin solution and shaken at 37°C for 2 h. The skin was then washed 4 times with purified water, 20 min / time.

[0064] (3) The substrate treated in step (2) was placed in a 1 M NaOH solution, shaken at 25° C. for 1 h, and then washed with purified water three times, 10 min each time;

[0065] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 25° C. for 1 h, and then ultrasonically washing with a 0.01 M PBS solution for 3 times, 10 min each time;

[0066] (5) placing the substrate treated in step (4) in a 1% TritonX-100 solution, shaking at 25°C for 4 h, and then ultrasonically washing it with a 0.01M PBS solution for 5 times, 10 min / time, and then washing it with purified water for 3 times, 20 min / time;

[0067] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0068] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and glycine evenly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids is 100:8:8:2:1;

[0069] (8) The syringe is placed in an insulated box with a layer, and dry ice or liquid nitrogen is placed in the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 25 KGy and the irradiation time is 8 h.

[0070] Example 5

[0071] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0072] (1) fresh pig skin is selected, fat and minced meat on the surface are removed, blood is removed by soaking in purified water, the skin is washed, cut into pieces, and crushed in a tissue grinder. The crushed tissue is washed with deionized water, soaked in 75% ethanol for 20 hours, washed with deionized water, and then the crushed tissue is placed in deionized water. Pepsin is added with 0.005N hydrochloric acid, and the amount of pepsin added is 1.5% of the weight of the pig skin. After enzymatic hydrolysis at 23°C for 60 hours, the supernatant is collected by centrifugation, adjusted to neutral with 1M NaOH, salted out with saturated NaCl for 8 hours, centrifuged, and the precipitate is dialyzed against deionized water to obtain collagen gel;

[0073] (2) Fresh pig skin was selected and removed using a DQ-2A 400×900 electric skin remover. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.5 mm was placed in a 0.32% trypsin solution and shaken at 35°C for 3 h. The skin was then washed with purified water for 5 times, 20 min / time.

[0074] (3) The substrate treated in step (2) was placed in a 0.5 M NaOH solution at 23° C. and shaken for 1.5 h, and then washed with purified water 4 times, 10 min each time;

[0075] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 23° C. for 1.5 h, and then ultrasonically washing with a 0.005 M PBS solution for 4 times, 10 min per time;

[0076] (5) placing the substrate treated in step (4) in a 2.5% TritonX-100 solution, shaking at 23°C for 5 h, and then ultrasonically washing it with a 0.005M PBS solution for 6 times, 10 min / time, and then washing it with purified water for 4 times, 20 min / time;

[0077] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0078] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and valine uniformly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and amino acids is 100:5:5:1:0.5;

[0079] (8) The syringe is placed in an insulated box with a sandwich layer, and dry ice or liquid nitrogen is placed in the sandwich layer of the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 15 KGy and the irradiation time is 10 h.

[0080] Comparative Example 1

[0081] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0082] (1) Select fresh cowhide, 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 24 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.01N hydrochloric acid, and the amount of pepsin added is 3% of the weight of the cowhide. After enzymatic hydrolysis at 25°C for 48 hours, centrifuge and collect the supernatant, adjust to neutral with 2M NaOH, salt out with saturated NaCl for 10 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0083] (2) Fresh cowhide was selected and the skin was removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.8 mm was placed in a 0.125% trypsin solution and shaken at 37°C for 2 h. The skin was then washed 4 times with purified water, 20 min / time.

[0084] (3) The substrate treated in step (2) was placed in a 1 M NaOH solution, shaken at 25° C. for 1 h, and then washed with purified water three times, 10 min each time;

[0085] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 25° C. for 1 h, and then ultrasonically washing with a 0.01 M PBS solution for 3 times, 10 min each time;

[0086] (5) placing the substrate treated in step (4) in a 1% TritonX-100 solution, shaking at 25°C for 4 h, and then ultrasonically washing it with a 0.01M PBS solution for 5 times, 10 min / time, and then washing it with purified water for 3 times, 20 min / time;

[0087] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0088] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and glycine uniformly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, acellular dermal matrix particles, glycerol, and amino acids is 100:16:2:1;

[0089] (8) The syringe is placed in an insulated box with a layer, and dry ice or liquid nitrogen is placed in the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 25 KGy and the irradiation time is 8 h.

[0090] Comparative Example 2

[0091] A method for preparing an active injection gel that can withstand irradiation sterilization is carried out according to the following steps:

[0092] (1) Select fresh cowhide, 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 24 hours, wash with deionized water, and then place the crushed tissue in deionized water. Add pepsin with 0.01N hydrochloric acid, and the amount of pepsin added is 3% of the weight of the cowhide. After enzymatic hydrolysis at 25°C for 48 hours, centrifuge and collect the supernatant, adjust to neutral with 2M NaOH, salt out with saturated NaCl for 10 hours, centrifuge, and dialyze the precipitate against deionized water to obtain collagen gel;

[0093] (2) Fresh cowhide was selected and the skin was removed using a DQ-2A 400×900 electric skin extractor. The epidermis, papillary dermis and subcutaneous fat were removed. The remaining middle dermis reticular layer with a thickness of 0.8 mm was placed in a 0.125% trypsin solution and shaken at 37°C for 2 h. The skin was then washed 4 times with purified water, 20 min / time.

[0094] (3) The substrate treated in step (2) was placed in a 1 M NaOH solution, shaken at 25° C. for 1 h, and then washed with purified water three times, 10 min each time;

[0095] (4) placing the substrate treated in step (3) in a 0.1% SDS solution, shaking at 25° C. for 1 h, and then ultrasonically washing with a 0.01 M PBS solution for 3 times, 10 min each time;

[0096] (5) placing the substrate treated in step (4) in a 1% TritonX-100 solution, shaking at 25°C for 4 h, and then ultrasonically washing it with a 0.01M PBS solution for 5 times, 10 min / time, and then washing it with purified water for 3 times, 20 min / time;

[0097] (6) placing the matrix treated in step (5) into a material storage tube and grinding it in a grinder, wherein the grinder is a SPEX6670 cryo-grinder, and the parameters are set as follows: precooling for 10 minutes, number of cycles is 3, running time is 2 minutes, and cooling time is 2 minutes; after grinding, the material storage tube is taken out and opened to obtain acellular dermal matrix particles;

[0098] (7) mixing collagen gel, acellular dermal matrix particles, bacterial cellulose, glycerol, and glycine evenly, decompressing and degassing, and injecting into a syringe; the mass ratio of the collagen gel, bacterial cellulose, glycerol, and amino acid is 100:16:2:1;

[0099] (8) The syringe is placed in an insulated box with a layer, and dry ice or liquid nitrogen is placed in the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 25 KGy and the irradiation time is 8 h.

[0100] Experimental example:

[0101] (1)DSC determination: 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 The test object is the injection gel prepared in step (7) of Example 1; the injection gel is treated with liquid nitrogen or dry ice at low temperature and then irradiated, and the irradiation dose is 25 KGy.

[0102] From the DSC test results, it can be seen that ( 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 was <4°C, which was within the error range of the instrument. It can be basically determined that the gel did not denature before and after irradiation.

[0103] (2) Injection performance: Injection force was measured using INSTRON4204 at room temperature, displacement rate of 5 mm / min, termination condition of L = 200 N, and load size when uniform injection was achieved. The ideal injection force should be less than 50 N. Each group of experiments was measured 5 times in parallel and the average value was taken. SPSS24.0 software was used for statistical analysis. The quantitative data results were used The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used for normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05.

[0104] The measurement results are shown in Table 1:

[0105] Table 1

[0106]

[0107] Note: * represents P < 0.05 compared with Example 1.

[0108] (3) Comparison of gel push injection: The injection gel prepared in step (7) of Example 1 was placed in 10 1 mL syringes with 27G needles, and 5 of them were randomly selected and placed in an incubator with an interlayer, and dry ice was placed in the interlayer for irradiation. The irradiation dose was 25 KGy. After irradiation, push injection experiments were performed before and after irradiation to observe the state of the gel during the push injection process and whether the gel push injection was not smooth or intermittent due to the denaturation of the gel due to irradiation.

[0109] The results are as follows Figure 3As shown, before and after irradiation, the injection gel can maintain a uniform, smooth and continuous colloid during the push injection process. No discontinuity occurs and the gel is denatured.

[0110] (4) In vivo experiment: In order to observe the biocompatibility of the gel and the induction of tissue regeneration, an in vivo subcutaneous implantation test was performed. SD rats were used as the experimental model, anesthetized with 0.3% pentobarbital, and subcutaneously implanted with 1 mL of the injection gel prepared in Example 1 as the experimental group. Hyaluronic acid was used as the control group. Samples were taken on the 28th day, and the tissue containing the implant was excised and fixed in a fixative. As pathological specimens, HE and Masson staining were performed and analyzed.

[0111] On day 28, HE staining results showed that ( Figure 4 ), the skin tissue structure of the control group was severely abnormal, and a large number of inflammatory cells were seen in the granulation tissue. A small number of inflammatory cells were seen in the dermis of the experimental group, and the fibroblasts and new blood vessels were more abundant than those in the control group. The results of Masson's trichrome staining showed that ( Figure 5 ), the collagen density of the experimental group was higher than that of the control group, indicating that the experimental group had a stronger ability to stimulate and promote collagen regeneration after implantation.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing an active injectable 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°C for 40-60 hours. Centrifuge to collect the supernatant, adjust to neutral with 1-3M NaOH, salt out with saturated NaCl for 8-12 hours, centrifuge, and dialyze the precipitate against 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 matrix 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 substrate treated in step (4) in a TritonX-100 solution, shaking at 23-27° C. for 3-5 h, and then ultrasonically washing it with a 0.005-0.015 M PBS solution for 4-6 times, 10 min / time, and then washing it with purified water for 2-4 times, 20 min / 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 inject into a syringe; (8) The syringe is placed in an insulated box with a sandwich layer, and dry ice or liquid nitrogen is placed in the sandwich layer of the insulated box. After placement, the insulated box is closed and Co60 irradiation is performed. The irradiation dose is 15-35 KGy and the irradiation time is 6-10 h.

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, birds, 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 is 3, running time is 2 min, and cooling time is 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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