Preparation method of sterile active collagen for tissue regeneration

Abstract

The invention relates to preparation of sterile active collagen capable of being used for tissue regeneration, belongs to the field of tissue regeneration of medical biological materials, and particularly relates to a method for preparing collagen by using animal tissues as raw materials, and the technical scheme comprises the following steps: raw material pretreatment and disinfection: removing foreign matters on the surface of the raw materials; carrying out decellularization and degreasing pretreatment by using high-permeability salt, dilute alkali liquor and an organic solvent / decontaminating agent, then disinfecting by using a disinfectant / preservative, removing surface moisture, and carrying out vacuum packaging and freezing; carrying out enzymolysis on the prepared raw materials to prepare a collagen crude extract; and sterilizing the collagen crude extract, and adjusting the pH value to obtain the sterile active collagen. Immunogenic substances such as a degerming aid, endotoxin and terminal peptide are effectively removed, the prepared collagen is aseptically provided, a triple helix natural active structure is maintained, the tissue regeneration effect is good, and the collagen has a good clinical application prospect.

Description

Technical Field

[0001] The invention relates to the field of medical material preparation, and in particular to a method for preparing sterile active collagen that can be used for tissue regeneration. Background Art

[0002] As an important biomaterial, collagen is widely used in medical dressings, medical cosmetic fillings, and tissue regeneration scaffolds because of its low immunogenicity, degradability, good tissue compatibility, and ability to promote tissue regeneration. Studies have shown that collagen dressings made of collagen solution and non-woven fabrics can provide a moist environment for the skin, repair the skin barrier, and promote wound healing. Collagen extracted from tendon tissue can be used as a cell scaffold to induce cartilage regeneration and repair articular cartilage defects. Even collagen 3D printing technology can manufacture tissue substitutes and allogeneic organs. In recent years, the market demand for medical collagen has maintained a high growth trend.

[0003] All collagens interact with some protein families, such as cell surface receptors, through triple helical domains, thereby mediating a series of cellular regulatory behaviors, such as cell adhesion and migration, hemostasis and immune function. Collagen with a triple helical structure of more than 300kD is usually called active collagen. Compared with recombinant collagen, collagen extracted from animal tissues has a more complete triple helical structure and is more widely used in clinical practice. However, the aseptic preparation process of animal-derived collagen often changes the structural integrity of collagen and its receptor binding properties, greatly reducing its biological activity. How to obtain sterile collagen, and how to process it without destroying the active structure of collagen so that the material reaches medical grade is a major challenge currently facing the industry.

[0004] CN 114306719 A discloses a collagen dressing and its preparation method, wherein an antioxidant is added for low temperature irradiation. CN 114634651 A discloses a sterile porous collagen repair gel and its preparation method, wherein glycerol and preservative are added for low temperature irradiation. The aseptic process introduces new substances, and repeated freezing and thawing causes a certain degree of damage to the protein structure. Summary of the invention

[0005] In view of the problems raised by the above background technology, the present invention provides a method for preparing sterile active collagen that can be used for tissue regeneration.

[0006] The process controls the cleanliness of the environment and combines biochemical methods to destroy the bacterial structure and achieve the purpose of sterilization. The process does not involve factors that cause protein denaturation, such as freezing, high temperature and radiation, and well maintains the triple helix active structure of collagen. At the same time, ultrafiltration purification removes processing aids and endotoxins, greatly reducing the immunogenicity of the material. The prepared collagen has a relatively simple composition and does not contain additives such as protective agents, antioxidants, and preservatives. It can be flexibly used for a variety of medical purposes such as tissue regeneration according to the purpose of use.

[0007] Specifically, the present invention provides the following technical solutions: A method for preparing sterile active collagen that can be used for tissue regeneration, characterized in that it comprises the following steps: A1. Pretreatment and disinfection of raw materials: remove foreign matter from the surface of raw materials, use hypertonic salt, dilute alkali solution, organic solvent / detergent for decellularization and degreasing pretreatment, then use disinfectant / preservative for pre-sterilization, remove surface moisture, vacuum pack and freeze; A2, slicing and enzymatically hydrolyzing the raw material obtained in step A1 to obtain a crude collagen extract; A3. Sterilize the crude collagen extract obtained in step A2 and adjust the pH to between 5 and 8 to obtain sterile active collagen.

[0008] Furthermore, the preparation method of sterile active collagen that can be used for tissue regeneration is characterized in that in the step A1, the raw materials are fresh animal tendons, skins, bones and other collagen-rich tissues; the hypertonic salt treatment is to treat with a sodium chloride or potassium chloride solution with a mass fraction of 15-30% for 30-120 minutes, repeatedly wash with injection water for 3 times and drain the water; the dilute alkali solution treatment is to treat with a potassium hydroxide, sodium hydroxide or sodium bicarbonate solution with a mass fraction of 0.01-0.1% for 2-10 minutes, repeatedly wash with injection water for 3 times and drain the water; the organic solvent / detergent treatment is to treat with a mass fraction of 0.05%-1 % polyethylene glycol octylphenyl ether-100, tributyl phosphate, EDTA-Na2, dodecyltrimethylammonium bromide, one or more of each for 2-10 minutes, rinse with injection water for 3 times and drain the water; pre-sterilization is to use 75% ethanol solution by mass, 0.1-0.5% hypochlorous acid solution, 2.5-5% hydrogen peroxide solution, 0.01%-2% peroxyethanedioic acid solution, two or more of each for 1-10 minutes, rinse with injection water for 3 times and drain the water; the whole pretreatment process including the disinfection step shall be carried out at a cleanliness level of not less than 100,000, and the solution used shall be prepared with injection water.

[0009] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that in step A2, pepsin is used for dynamic stirring enzymolysis at 2~10°C under acidic conditions for 40~120h, the acid used under the acidic conditions is 0.1~1.0M phosphoric acid and 0.1~0.5M citric acid or acetic acid, the mass volume ratio of bovine Achilles tendon: pepsin: acid is 1g:0.01g:60ml, and the entire operation process must be carried out at a cleanliness level of not less than Class 10,000.

[0010] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that in step A2, 0.05% to 1% by mass of polyethylene glycol octylphenyl ether-100 or diethylene glycol mono[(1,1,3,3-tetramethylbutyl)phenyl] ether, 0.01% to 0.5% of EDTA-Na2, 0.1% to 1% of polysorbate 80 and 0.5% to 1.5% of calcium chloride are added to the enzymatic solution 2 to 8 hours before the end of the enzymatic hydrolysis as sterilization aids.

[0011] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that, in step A3, the crude collagen extract obtained in step A2 needs to be homogenized under high pressure before being sterilized, and ultrafiltration is performed after the solution is clarified.

[0012] Furthermore, the method for preparing sterile active collagen for tissue regeneration is characterized in that in step A3, the high-pressure homogenization process parameters of the crude collagen extract are 200-1000 bar, 2-15° C., and homogenization for 20-90 min. The high-pressure homogenizer is electric or pneumatic, and the homogenizing valve is a plunger type or a microfluidic valve core; or a high-pressure homogenizer with a similar homogenization principle is used.

[0013] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that, in step A3, a sterile filter membrane with a relative molecular mass of 50KD~100KD is used in the ultrafiltration process; the solvent in the collagen solution is replaced by 3~5 times the volume with a 0.01~0.1mol / L dilute acid solution; the solution volume is concentrated to 1 / 2~1 / 20 of the original volume in the last replacement; and the dilute acid solution is prepared into a solution using one or more of hydrochloric acid, acetic acid, citric acid, and phosphate.

[0014] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that in step A3, the pH regulator can be 1% to 20% sodium hydroxide or potassium hydroxide, and the content of the obtained sterile active collagen is 6 to 100 mg / ml.

[0015] Furthermore, the method for preparing sterile active collagen that can be used for tissue regeneration is characterized in that all operation processes in step A3 need to be carried out in a class 100 laminar flow environment, and the instruments used should be sterilized in advance.

[0016] Furthermore, the method for preparing the sterile active collagen that can be used for tissue regeneration is characterized in that the sterile active collagen can be used in the field of tissue regeneration medicine.

[0017] The present invention provides a method for preparing sterile active collagen that can be used for tissue regeneration. The present invention has the following advantages: 1. The present invention provides a sterile active collagen that can be used for tissue regeneration. The preparation method controls microorganisms throughout the entire operation process, uses a sterilization aid to destroy the membrane structure of the remaining bacteria, and uses a high-pressure homogenization operation to further break up the bacterial cells and kill them. In the process of aseptic operation of collagen, compared with the final irradiation sterilization, the natural active structure of the triple helix is ​​maintained to the greatest extent. 2. The preparation method of the present invention can remove sterilization aids, endotoxins, and terminal peptides and other substances that cause immunogenicity. The prepared collagen has good biocompatibility and low immunogenicity, and can promote cell migration and tissue regeneration. 3. Compared with traditional methods such as multiple salting out, centrifugation, chromatography, etc., the preparation method of the present invention simplifies the process steps, reduces production operation difficulties, and is conducive to industrial production. 4. The collagen prepared by the present invention introduces calcium and phosphorus ions, has good tissue regeneration and repair effects, can be compounded with hyaluronic acid, hydroxyapatite, chondroitin sulfate, chitosan, chitosan, trehalose, growth factors, etc. to make biological scaffolds, and can also be used as a sustained-release carrier for drugs, growth factors, etc., and has broad application prospects in different medical fields. BRIEF DESCRIPTION OF THE DRAWINGS In order to explain the present invention more clearly, the specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0019] Figure 1 Collagen electrophoresis diagram. Figure 2 This is a transmission electron microscopy diagram of the triple helix structure. Figure 3 This is a diagram of collagen cell migration. DETAILED DESCRIPTION The technical solution of the present invention is clearly and completely described below in conjunction with specific implementation methods. It is obvious that the specific embodiments described are only part of the embodiments of the present invention, not all of the embodiments, and do not constitute a limitation on the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all reagents used in the following specific examples are commercially available. Example

[0022] The present embodiment provides a method for preparing sterile active collagen that can be used for tissue regeneration, the method comprising the following steps: A1. In a 100,000-class cleanroom, take 100g of bovine Achilles tendon to remove foreign matter on the surface, treat it with 15% sodium chloride solution for 60 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.1% sodium hydroxide by mass for 2 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.1% polyethylene glycol octylphenyl ether-100, 0.3% tributyl phosphate, 0.1% EDTA-Na2, 0.1% dodecyl trimethyl ammonium bromide for 5 minutes each, wash it repeatedly with injection water for 3 times each time and drain the water; treat it with 75% ethanol solution by mass for 2 minutes, 0.1% hypochlorous acid solution for 5 minutes, 2.5% hydrogen peroxide solution for 2 minutes, 0.2% peroxyethanedioic acid solution for 2 minutes, wash it repeatedly with injection water for 3 times each time and drain the water. After removing the surface moisture, vacuum pack and freeze. The solution used is prepared with injection water.

[0023] A2. In a Class 10,000 cleanroom, cut the bovine Achilles tendon obtained in step A1 into slices of about 1 mm, add 2L 0.1M phosphoric acid and 4L 0.5M acetic acid mixed solution containing 1g pepsin, and perform enzymolysis under dynamic stirring at 2-10°C. After 115h of enzymolysis, add 0.1% diethylene glycol mono[(1,1,3,3-tetramethylbutyl)phenyl]ether, 0.01% EDTA-Na2, 0.05% polysorbate 80, and 0.5% calcium chloride.

[0024] A3. Under a Class 100 environment, the crude collagen extract from step A2, which was hydrolyzed for 120 hours, was homogenized for 30 minutes at 2-15°C using a high-pressure homogenizer at 500 bar, and ultrafiltered using a sterile filter membrane with a relative molecular mass of 100KD and a 0.01mol / L hydrochloric acid solution. The volume of the solvent in the collagen solution was replaced by 5 times, and the volume of the solution was concentrated to 1 / 12 of the original volume in the last replacement. The pH of the solution was adjusted to 7.6 with 10% sodium hydroxide, and the content of the sterile active collagen obtained was 35mg / ml. Example

[0025] The present embodiment provides a method for preparing sterile active collagen that can be used for tissue regeneration, the method comprising the following steps: A1. In a 100,000-class cleanroom, take 100g of bovine Achilles tendon to remove foreign matter on the surface, treat it with 30% sodium chloride solution for 120 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.01% sodium hydroxide by mass for 5 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.1% polyethylene glycol octylphenyl ether-100 by mass for 10 minutes, 0.3% tributyl phosphate for 8 minutes, and 0.1% EDTA-Na2 for 5 minutes, wash it repeatedly with injection water for 3 times each time and drain the water; treat it with 0.1% sodium hypochlorite solution by mass for 2 minutes, 2.5% hydrogen peroxide solution for 5 minutes, and 0.1% peroxyethanedioic acid solution for 5 minutes, wash it repeatedly with injection water for 3 times each time and drain the water. After removing the surface moisture, vacuum pack and freeze. The solution used is prepared with injection water.

[0026] A2. In a Class 10,000 cleanroom, cut the bovine Achilles tendon obtained in step A1 into slices of about 1 mm, add 2L 0.1M phosphoric acid and 4L 0.5M citric acid mixture containing 1g pepsin, and perform enzymolysis under dynamic stirring at 2-10°C. After 60h of enzymolysis, add 0.1% polyethylene glycol octylphenyl ether-100, 0.01% EDTA-Na2, 0.05% polysorbate 80 and 0.5% calcium chloride.

[0027] A3. Under a Class 100 environment, the crude collagen extract from step A2, which was hydrolyzed for 68 hours, was homogenized for 40 minutes at 2-15°C using a high-pressure homogenizer at 400 bar, and ultrafiltered using a sterile filter membrane with a relative molecular mass of 100 KD and a 0.1 mol / L citric acid solution. The volume of the solvent in the collagen solution was replaced by 3 times, and the volume of the solution was concentrated to 1 / 4 of the original volume in the last replacement. The pH of the solution was adjusted to 6 using 5% sodium hydroxide, and the content of the sterile active collagen obtained was 10 mg / ml. Example

[0028] The present embodiment provides a method for preparing sterile active collagen that can be used for tissue regeneration, the method comprising the following steps: A1. In a 100,000-class cleanroom, take 100g of bovine Achilles tendon to remove foreign matter on the surface, treat it with 20% sodium chloride solution for 90 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.1% sodium hydroxide by mass for 2 minutes, wash it repeatedly with injection water for 3 times and drain the water; treat it with 0.1% polyethylene glycol octylphenyl ether-100, 0.3% tributyl phosphate, and 0.1% dodecyl trimethyl ammonium bromide for 5 minutes each, wash it repeatedly with injection water for 3 times each time and drain the water; treat it with 75% ethanol solution by mass for 2 minutes, 0.1% hypochlorous acid solution for 5 minutes, and 0.2% peroxyethanedioic acid solution for 2 minutes, wash it repeatedly with injection water for 3 times each time and drain the water. After removing the surface moisture, vacuum pack and freeze. The solution used is prepared with injection water.

[0029] A2. In a Class 10,000 cleanroom, cut the bovine Achilles tendon obtained in step A1 into slices of about 1 mm, add 2L 0.1M phosphoric acid and 4L 0.5M acetic acid mixed solution containing 1g pepsin, and perform enzymolysis under dynamic stirring at 2-10°C. After 46h of enzymolysis, add 0.1% diethylene glycol mono[(1,1,3,3-tetramethylbutyl)phenyl]ether, 0.01% EDTA-Na2, 0.05% polysorbate 80 and 0.5% calcium chloride.

[0030] A3. Under a Class 100 environment, the collagen crude extract from step A2, which was hydrolyzed for 48 hours, was homogenized for 20 minutes using a high-pressure homogenizer at 800 bar and 2-15°C, and ultrafiltered using a sterile filter membrane with a relative molecular mass of 100KD and a 0.1mol / L hydrochloric acid solution. The volume of the solvent in the collagen solution was replaced by 5 times, and the volume of the solution was concentrated to 1 / 20 of the original volume in the last replacement. The pH of the solution was adjusted to 7.0 with 20% sodium hydroxide, and the content of the sterile active collagen obtained was 85mg / ml.

[0031] Comparative Example 1: The collagen preparation method of this comparative example is based on the operating steps of Example 1, except that the raw material pretreatment and disinfection operations in step A1 are not performed.

[0032] Comparative Example 2: The collagen preparation method of this comparative example is based on the operating steps of Example 1, except that step A3 is not performed under a cleanliness level of 10,000.

[0033] Comparative Example 3: The collagen preparation method of this comparative example is based on the operating steps of Example 1, except that high-pressure homogenization is not performed in step A2.

[0034] Comparative Example 4: The collagen preparation method of this comparative example is based on the operating steps of Example 1, except that ultrafiltration operation is not performed in step A3.

[0035] Effect Example 1 Sterility and Endotoxin Detection The collagen products obtained in Examples 1 to 3 and Comparative Examples 1 to 4 described above were tested for sterility and endotoxin, and the experimental data are as follows (Table 1): Table 1 Sterility and endotoxin test results As can be seen from Table 1, Examples 1 to 3 can obtain sterile negative collagen with lower endotoxin levels than Comparative Examples 1 to 4. Comparative Example 1 did not perform the raw material pretreatment and disinfection operations in step A1. Due to the large number of microorganisms initially carried, the endotoxin content of the collagen was high and could not be used in the medical field. In Comparative Example 2, step A3 was not produced at a cleanliness level of 10,000, resulting in a sterile positive collagen product with a high endotoxin level. Comparative Example 3 did not perform the high-pressure homogenization operation in step A3. Although the ultrafiltration operation in step A3 removed endotoxins, the bacteria in the collagen product were not killed, resulting in sterile positive. Comparative Example 4 did not perform the ultrafiltration operation in step A3. Although it was sterile negative, the endotoxins produced during the treatment process were not completely removed.

[0036] Effect Example 2 SDS-PAGE Detection of Collagen Products The electrophoresis test method is used to test and obtain the electrophoresis diagram (such as Figure 1 as shown).

[0037] Said Figure 1 1 is the collagen extracted in comparative example 1 of the method of the present invention, 2 is the collagen extracted in comparative example 2 of the method of the present invention, 3 is the collagen extracted in comparative example 3 of the method of the present invention, 4 is the collagen extracted in comparative example 4 of the method of the present invention, 5 is the collagen extracted in example 1 of the method of the present invention, 6 is the collagen extracted in example 2 of the method of the present invention, 7 is the collagen extracted in example 3 of the method of the present invention, and 8 is a collagen reference substance.

[0038] The results are as follows: the collagen extracted by the present invention is electrophoresed with the standard Figure 1 The original triple helix active structure is maintained.

[0039] Effect Example 3 Transmission electron microscopy detection of collagen products Transmission electron microscopy was performed on the above-mentioned Examples 1 to 3 and Comparative Examples 1 to 4. The results are as follows: Figure 2 For example, light and dark alternating bands can be observed, indicating that the collagen products extracted by the present invention all maintain the triple helix active structure.

[0040] Effect Example 4 Collagen Product Cell Migration Experiment Cell migration is common in physiological and pathological processes such as injury repair, cell differentiation, embryonic development, and tumor metastasis. A cell migration experiment was conducted on the collagen products of Examples 1 to 3 described above to detect the migration of osteoblasts in the collagen products for 24 hours. Figure 3 In the figure, a, b, and c are 24-hour cell migration images of the blank cell group, d is the 24-hour cell migration image of the collagen in Example 1, e is the 24-hour cell migration image of the collagen in Example 2, f is the 24-hour cell migration image of the collagen in Example 3, and h, i, and j are the 24-hour cell migration images of the positive control group.

[0041] The results showed that the collagen product of the embodiment of the present invention had a good cell migration effect, and the average cell migration rate (68.03%) was greater than that of the positive control group (51.52%).

[0042] In summary, the collagen extracted by the collagen preparation method of the present invention is sterile and negative, maintains its natural active structure, has a low endotoxin level and low immunogenicity, can well guide cell migration, and has important application value in the field of biomedical material tissue regeneration.

[0043] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing sterile active collagen that can be used for tissue regeneration, characterized in that: The technical solution comprises the following steps: A1. Pretreatment and disinfection of raw materials: remove foreign matter from the surface of raw materials, use hypertonic salt, dilute alkali solution, organic solvent / detergent for decellularization and degreasing pretreatment, then use disinfectant / preservative for pre-sterilization, remove surface moisture, vacuum pack and freeze; A2, slicing and enzymatically hydrolyzing the raw material obtained in step A1 to obtain a crude collagen extract; A3. Sterilize the crude collagen extract obtained in step A2 and adjust the pH to between 5 and 8 to obtain sterile active collagen.

2. A method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: In the step A1, the raw materials are fresh animal tendons, skins, bones and other collagen-rich tissues; the hypertonic salt treatment is to treat with a sodium chloride or potassium chloride solution with a mass fraction of 15-30% for 30-120 minutes, repeatedly wash with injection water for 3 times and drain the water; the dilute alkali solution treatment is to treat with a potassium hydroxide, sodium hydroxide or sodium bicarbonate solution with a mass fraction of 0.01-0.1% for 2-10 minutes, repeatedly wash with injection water for 3 times and drain the water; the organic solvent / detergent treatment is to treat with a mass fraction of 0.05 %~1% polyethylene glycol octylphenyl ether-100, tributyl phosphate, EDTA-Na2, dodecyl trimethyl ammonium bromide, one or more of each for 2~10 minutes, rinse with injection water for 3 times and drain the water; pre-sterilization is to use 75% ethanol solution, 0.1~0.5% hypochlorous acid solution, 2.5~5% hydrogen peroxide solution, 0.01%~2% peroxy oxalic acid solution, two or more of each for 1~10 minutes, rinse with injection water for 3 times and drain the water; The entire pretreatment process including the disinfection step must be carried out at a cleanliness level not lower than Class 100,000, and the solution used must be prepared with water for injection.

3. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: In the step A2, pepsin is used for dynamic stirring enzymolysis at 2-10° C. under acidic conditions for 40-120 hours, the acid used under the acidic conditions is 0.1-1.0 M phosphoric acid and 0.1-0.5 M citric acid or acetic acid, the mass volume ratio of bovine Achilles tendon: pepsin: acid is 1 g: 0.01 g: 60 ml, and the entire operation process must be carried out at a cleanliness level of not less than Class 10,000.

4. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: In the step A2, 2 to 8 hours before the end of the enzymatic hydrolysis, 0.05% to 1% by mass of polyethylene glycol octylphenyl ether-100 or diethylene glycol mono[(1,1,3,3-tetramethylbutyl)phenyl]ether, 0.01 to 0.5% of EDTA-Na2, 0.1% to 1% of polysorbate 80 and 0.5% to 1.5% of calcium chloride are added to the enzymatic hydrolysis solution as sterilization aids.

5. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: In the step A3, the crude collagen extract obtained in step A2 needs to be homogenized under high pressure before being sterilized, and ultrafiltration is performed after the solution is clarified.

6. A method for preparing sterile active collagen for tissue regeneration according to claim 5, characterized in that: In step A3, the parameters of high-pressure homogenization process of the crude collagen extract are 200-1000 bar, 2-15° C., and homogenization for 20-90 min. The high-pressure homogenizer is electric or pneumatic, and the homogenizing valve is a plunger type or a microfluidic valve core; or a high-pressure homogenizer with a similar homogenization principle is used.

7. A method for preparing sterile active collagen for tissue regeneration according to claim 5, characterized in that: In the step A3, a sterile filter membrane with a relative molecular mass of 50KD~100KD is used in the ultrafiltration process; the solvent in the collagen solution is replaced by 3~5 times the volume with a 0.01~0.1mol / L dilute acid solution; the volume of the solution is concentrated to 1 / 2~1 / 20 of the original volume in the last replacement; the dilute acid solution is a solution prepared by one or more of hydrochloric acid, acetic acid, citric acid, and phosphate.

8. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: In step A3, the pH adjuster may be 1% to 20% sodium hydroxide or potassium hydroxide, and the content of the obtained sterile active collagen is 6 to 100 mg / ml.

9. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: All operations in step A3 must be performed in a Class 100 laminar flow environment, and the instruments used must be sterilized in advance.

10. The method for preparing sterile active collagen for tissue regeneration according to claim 1, characterized in that: The sterilized active collagen can be used for tissue regeneration.

Images