Medical-grade tissue-like natural collagen as well as preparation method and application thereof

Abstract

The invention provides medical-grade tissue-like natural collagen as well as a preparation method and application thereof, and relates to a biomedical material. The invention provides a preparation method of medical grade tissue-like natural collagen, which comprises the following steps: removing subcutaneous tissue of animal skin, soaking in normal saline, sequentially carrying out degreasing treatment, unhairing treatment, swelling removal treatment, acid swelling treatment, ball milling, pH value adjustment to 7-7.4, solid-liquid separation, washing the obtained solid component with water, and drying to obtain the medical grade tissue-like natural collagen. The medical-grade tissue-like natural collagen is obtained. The medical grade tissue-like natural collagen prepared by the invention has good biocompatibility, proper biodegradability, good mechanical properties and excellent thermal stability and structural stability, is low in antigenicity and easy to preserve, can quickly and effectively stop bleeding and promote wound healing and repair, and has a wide application prospect. The material is an ideal material for biomedical materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a medical-grade tissue-like natural collagen and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant and widely distributed structural protein in animals. It is widely present in skin, Achilles tendon, cartilage, cornea and various connective tissues. Collagen makes the extracellular matrix have good mechanical properties and structural integrity, has the function of supporting organs and protecting the body, and plays a key role in maintaining the structure and physiological function of the body. The existence structure and morphology of collagen in living organisms have always been a hot topic in the field of collagen research. It has been confirmed that the existence of natural collagen in tissues is not the conventional collagen molecule level with a triple helix structure on the market, nor is it a simple single collagen fiber. Its complex physiological aggregation process in living organisms gives it a typical multi-level structural characteristic. The original collagen molecules with a triple helix structure synthesized by amino acids are arranged in parallel into bundles in a quarter-staggered manner, and are cross-linked by covalent bonds to form stable collagen microfibrils, which are then arranged in a step-by-step orderly manner to form protofibrils with a diameter of 50 to 200 nm and a length of 280 nm to several microns, and then aggregated into bundles to form collagen microfibrils. Multiple bundles of microfibrils can form protofibrils. The fibrils are assembled together to form fibers, and the fibers are further entangled to form collagen fiber bundles. Collagen fiber bundles form the basic tissue fiber structure of living organisms with a three-dimensional network by separating, interlacing, weaving and entanglement with each other. The basic form of natural collagen in tissues is collagen fiber bundles.

[0003] Collagen materials are widely used in the fields of tissue engineering, regenerative medicine, and implantable medical devices due to their excellent biocompatibility, cell affinity, and low antigenicity. At present, the processing and material preparation of collagen are mainly based on the acid-enzyme binding method. However, since the method involves the degradation process of natural collagen, it greatly destroys the aggregate structure of natural collagen, resulting in the application limitations of the prepared molecular-level collagen materials such as insufficient mechanical properties and poor thermal stability. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a medical-grade tissue-like natural collagen and its preparation method and application. The medical-grade tissue-like natural collagen prepared by the preparation method provided by the present invention has excellent mechanical properties, thermal stability and hemostatic properties, and degrades slowly, which can significantly promote tissue regeneration and repair and reduce the formation of scars.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing medical-grade tissue-like natural collagen, comprising the following steps:

[0007] removing subcutaneous tissue of the animal skin and soaking it in physiological saline to obtain pretreated animal skin;

[0008] Degreasing the pretreated animal skin to obtain defatted animal skin;

[0009] Dehairing the defatted animal skin to obtain a dehaired animal skin;

[0010] performing a deswelling treatment on the depilated animal skin to obtain a deswelling animal skin;

[0011] subjecting the de-swollen animal skin to an acid swelling treatment to obtain an acid swollen animal skin;

[0012] The acid-swollen animal skin is crushed, the pH value is adjusted to 7-7.4, the solid-liquid separation is carried out to obtain a solid component, the solid component is washed with water and then dried to obtain medical-grade tissue-like natural collagen.

[0013] Preferably, the degreasing treatment comprises: adding a degreasing agent and water to the pretreated animal skin to perform degreasing treatment;

[0014] The mass ratio of the pretreated animal skin to the degreasing agent is 1:0.01-0.04;

[0015] The degreasing agent includes one or more of alkyl polyglycosides, peregrin, washing powder, sodium lauryl sulfate and fatty alcohol polyoxyethylene ether;

[0016] The mass volume ratio of the pretreated animal skin to water is 1kg:1.5-3L;

[0017] The degreasing treatment is performed at a temperature of 30 to 37°C for 2 to 5 hours;

[0018] The degreasing treatment further includes water washing.

[0019] Preferably, the depilatory treatment comprises: adding the defatted animal skin, water, polyoxyethylene ether and ferrous salt to perform a first depilatory treatment, adding alcohol amine to perform a second depilatory treatment, adding an alkaline agent to perform a third depilatory treatment, adding an oxidant to perform a fourth depilatory treatment, and adding a dilute alkaline agent aqueous solution to perform a fifth depilatory treatment;

[0020] The mass volume ratio of the defatted animal skin and water is 1kg:1-2L;

[0021] The mass ratio of the pretreated animal skin to the polyoxyethylene ether is 1:0.3-0.6;

[0022] The mass ratio of the pretreated animal skin to the ferrous salt is 1:0.3-0.6;

[0023] The first hair removal treatment lasts for 90 to 150 minutes;

[0024] The mass ratio of the pretreated animal skin to the alcohol amine is 1:0.01-0.03;

[0025] The second hair removal treatment lasts for 10 to 20 minutes;

[0026] The mass ratio of the pretreated animal skin to the alkaline reagent is 1:0.035-0.045;

[0027] The third hair removal treatment lasts for 15 to 25 minutes;

[0028] The mass ratio of the pretreated animal skin to the oxidant is 1:0.06-0.08;

[0029] The fourth hair removal treatment lasts for 3 to 4 hours;

[0030] The mass fraction of the dilute alkaline reagent aqueous solution is 0.3-0.8%; the time of the fifth depilatory treatment is 4-6 hours;

[0031] The depilatory treatment further comprises washing with water.

[0032] Preferably, the polyoxyethylene ether includes fatty alcohol polyoxyethylene ether and / or nonylphenol polyoxyethylene ether;

[0033] The ferrous salt includes ferrous sulfate and / or ammonium ferrous sulfate;

[0034] The alcohol amine includes one or more of triethanolamine, monoethanolamine and diethanolamine;

[0035] The alkaline agent includes alkali metal hydroxide and / or alkali metal carbonate;

[0036] The oxidant includes one or more of hydrogen peroxide, sodium perborate and sodium percarbonate;

[0037] The alkaline agent in the dilute alkaline agent aqueous solution includes alkali metal hydroxide and / or alkali metal carbonate.

[0038] Preferably, the deswelling treatment comprises: adding a first portion of water and an inorganic salt to the dehaired animal skin for a first deswelling treatment, adding a second portion of water, adjusting the pH value to 4 to 4.5, adding acid lipase for a second deswelling treatment, and adding acid protease for a third deswelling treatment;

[0039] The mass volume ratio of the pretreated animal skin to the first portion of water is 1 kg: 0.8 to 1 L;

[0040] The inorganic salt includes one or more of sodium bisulfite, ammonium sulfate, ammonium chloride and ammonium acetate; the mass of the inorganic salt accounts for 0.05-0.2% of the mass of the pretreated animal skin;

[0041] The temperature of the first deswelling treatment is 28-35°C, the time is 50-60 minutes, and the end point pH value is 8-8.5;

[0042] The mass volume ratio of the pretreated animal skin to the second part of water is 1 kg: 1 to 4 L;

[0043] The mass of the acid lipase accounts for 0.1-0.3% of the mass of the pretreated animal skin; the enzyme activity of the acid lipase is 50,000-60,000 units / g;

[0044] The second deswelling treatment is performed at a temperature of 28 to 35°C for 15 to 20 minutes;

[0045] The mass of the acidic protease accounts for 0.1-0.3% of the mass of the pretreated animal skin; the enzyme activity of the acidic protease is 80,000-100,000 units / g;

[0046] The temperature of the third deswelling treatment is 38-42°C and the time is 2-5 hours;

[0047] The deswelling treatment further includes water washing.

[0048] Preferably, the acid swelling treatment comprises: adding water to the de-swollen animal skin, adjusting the pH value to 2.8 to 3.0, stirring the acid swelling treatment and then standing the acid swelling treatment;

[0049] The mass volume ratio of the pretreated animal skin to water is 1 kg: 1 to 2 L;

[0050] The stirring acid swelling treatment time is 30 to 60 minutes;

[0051] The time of the static acid swelling treatment is 6 to 8 hours;

[0052] The acid swelling treatment further includes water washing.

[0053] Preferably, the pulverization includes ball milling, the ball-to-material ratio of the ball milling is 1:1-5, the rotation speed is 200-600r / min, and the time is 1-3h;

[0054] The drying comprises freeze drying;

[0055] The drying process also includes disinfection and sterilization.

[0056] Preferably, the animal skin comprises skin of one or more of pigs, cattle and sheep.

[0057] The present invention also provides medical-grade tissue-like natural collagen prepared by the preparation method described in the above technical solution.

[0058] The present invention also provides the use of the medical-grade tissue-like natural collagen described in the above technical solution in tissue engineering, regenerative medicine materials or implantable medical devices.

[0059] The present invention provides a method for preparing medical-grade tissue-like natural collagen, comprising the following steps: removing subcutaneous tissue of animal skin, soaking in physiological saline, sequentially performing degreasing treatment, depilation treatment, deswelling treatment, acid swelling treatment, crushing, first solid-liquid separation, adjusting the pH value of the obtained liquid component to 7-7.4, second solid-liquid separation, washing the obtained solid component with water and then drying, and obtaining medical-grade tissue-like natural collagen. The medical-grade tissue-like natural collagen prepared by the present invention has not undergone any degradation process of natural collagen, and its basic composition is the existence form of natural collagen in tissue-collagen fiber bundles, and also has the spatial structural characteristics of natural collagen in tissue, so that it can mimic natural tissue structure to a greater extent. The medical-grade tissue-like natural collagen prepared by the present invention has more excellent mechanical properties and excellent enzyme resistance / thermal stability and structural stability, low antigenicity and easier to preserve, excellent hemostatic performance, and its high strength and toughness can meet the diverse requirements of the body for material performance, and is an ideal collagen biomedical material. The medical-grade tissue-like natural collagen prepared by the present invention is beneficial to the survival, proliferation and differentiation of cells, and can significantly promote the regeneration and repair of tissues and reduce the generation of scars in wound and scar repair.

[0060] Moreover, the preparation method provided by the present invention has simple process, simple equipment, abundant raw materials, low cost, and is easy to scale up for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is an atomic force microscope (AFM) detection image of the medical-grade tissue-like natural collagen prepared in Example 1;

[0062] Figure 2 The MTT test results of collagen, antigen-free collagen aggregates prepared in Comparative Example 1, and medical-grade tissue-like natural collagen prepared in Example 2;

[0063] Figure 3 This is the result of 24h scratching of collagen;

[0064] Figure 4 This is a 24h scratch result diagram of the antigen-free collagen aggregate prepared in Comparative Example 1;

[0065] Figure 5 This is a 24-hour scratch result diagram of the medical-grade tissue-like natural collagen prepared in Example 2;

[0066] Figure 6 This is a HE-stained section of animal skin tissue of the medical-grade natural tissue collagen prepared in Example 2 (×100). DETAILED DESCRIPTION

[0067] The present invention provides a method for preparing medical-grade tissue-like natural collagen, comprising the following steps:

[0068] removing subcutaneous tissue of the animal skin and soaking it in physiological saline to obtain pretreated animal skin;

[0069] Degreasing the pretreated animal skin to obtain defatted animal skin;

[0070] Dehairing the defatted animal skin to obtain a dehaired animal skin;

[0071] performing a deswelling treatment on the depilated animal skin to obtain a deswelling animal skin;

[0072] subjecting the de-swollen animal skin to an acid swelling treatment to obtain an acid swollen animal skin;

[0073] The acid-swollen animal skin is crushed, the pH value is adjusted to 7-7.4, the solid-liquid separation is carried out to obtain a solid component, the solid component is washed with water and then dried to obtain medical-grade tissue-like natural collagen.

[0074] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0075] The invention removes subcutaneous tissue of animal skin and soaks it in physiological saline to obtain pretreated animal skin.

[0076] In the present invention, the animal skin preferably includes one or more animal skins of pigs, cattle and sheep; the animal skin is preferably fresh and traceable animal skin.

[0077] The present invention preferably first cleans the animal skin, soaks it in water, and then removes the subcutaneous tissue of the animal skin. In the present invention, the purpose of the cleaning is to remove dirt on the animal skin. In the present invention, the temperature of the water soaking is preferably room temperature, and the time of the water soaking is preferably 60 to 120 minutes, and in specific embodiments it can be 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes or 120 minutes. In the present invention, the method of removing the subcutaneous tissue of the animal skin preferably includes mechanical meat cutting; the subcutaneous tissue includes subcutaneous fat, surface muscle, blood vessels and fascia tissue.

[0078] In the present invention, the animal skin with subcutaneous tissue removed is preferably cut into pieces, washed with saline, and then soaked in saline. In the present invention, the size of the animal pieces obtained by cutting is preferably 5-10 mm×5-10 mm. In the present invention, the temperature of the saline soaking is preferably room temperature, and the time of the saline soaking is preferably 10-18 hours, and in specific embodiments, it can be 10 hours, 11 hours, 12 hours, 13 hours, 4 hours, 15 hours, 16 hours, 17 hours or 18 hours.

[0079] After obtaining the pretreated animal skin, the present invention performs a degreasing treatment on the pretreated animal skin to obtain the defatted animal skin.

[0080] In the present invention, the degreasing treatment preferably comprises: adding a degreasing agent and water to the pretreated animal skin to perform degreasing treatment;

[0081] In the present invention, the mass volume ratio of the pretreated animal skin to water is preferably 1kg:1.5-3L. In specific embodiments, it can be 1kg:1.5L, 1kg:1.6L, 1kg:1.7L, 1kg:1.8L, 1kg:1.9L, 1kg:2L, 1kg:2.1L, 1kg:2.2L, 1kg:2.3L, 1kg:2.4L, 1kg:2.5L, 1kg:2.6L, 1kg:2.7L, 1kg:2.8L, 1kg:2.9L or 1kg:3L.

[0082] In the present invention, the degreasing agent preferably includes one or more of alkyl polyglycosides, peregal, washing powder, sodium lauryl sulfate and fatty alcohol polyoxyethylene ether. In the present invention, the mass ratio of the pretreated animal skin to the degreasing agent is preferably 1:0.01-0.04, and in specific embodiments, it can be 1:0.01, 1:0.015, 1:0.02, 1:0.025, 1:0.03, 1:0.035 or 1:0.04.

[0083] In the present invention, the temperature of the degreasing treatment is preferably 30-37°C, and in specific embodiments it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C; the time of the degreasing treatment is preferably 2-5h, and in specific embodiments it can be 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h; the degreasing treatment is preferably carried out in a rotating drum; the rotation speed of the rotating drum is preferably 5-12r / min, and in specific embodiments it can be 5r / min, 6r / min, 7r / min, 8r / min, 9r / min, 10r / min, 11r / min or 12r / min.

[0084] In the present invention, the degreasing treatment preferably further comprises: washing the animal skin after the degreasing treatment to obtain defatted animal skin. In the present invention, the number of times of washing is preferably 3 to 5 times, more preferably 4 times.

[0085] After obtaining the defatted animal skin, the present invention performs a dehairing treatment on the defatted animal skin to obtain the dehaired animal skin.

[0086] In the present invention, the dehairing treatment preferably includes: adding the defatted animal skin, water, polyoxyethylene ether and ferrous salt for the first dehairing treatment, adding alcohol amine for the second dehairing treatment, adding alkaline reagent for the third dehairing treatment, adding oxidant for the fourth dehairing treatment, and adding dilute alkaline reagent aqueous solution for the fifth dehairing treatment. In the present invention, the temperature of the dehairing treatment is preferably room temperature, and the dehairing treatment is preferably carried out in a rotary drum; the rotation speed of the rotary drum is preferably 5 to 12 r / min, and in specific embodiments it can be 5 r / min, 6 r / min, 7 r / min, 8 r / min, 9 r / min, 10 r / min, 11 r / min or 12 r / min.

[0087] In the present invention, the mass volume ratio of the defatted animal skin and water is preferably 1kg:1-2L. In specific embodiments, it can be 1kg:1L, 1kg:1.1L, 1kg:1.2L, 1kg:1.3L, 1kg:1.4L, 1kg:1.5L, 1kg:1.6L, 1kg:1.7L, 1kg:1.8L, 1kg:1.9L or 1kg:2L.

[0088] In the present invention, the polyoxyethylene ether preferably includes fatty alcohol polyoxyethylene ether and / or nonylphenol polyoxyethylene ether. In the present invention, the mass ratio of the pretreated animal skin to the polyoxyethylene ether is preferably 1:0.3-0.6, and in specific embodiments it can be 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.55 or 1:0.6.

[0089] In the present invention, the ferrous salt preferably includes ferrous sulfate and / or ammonium ferrous sulfate. In the present invention, the mass ratio of the pretreated animal skin to the ferrous salt is preferably 1:0.3-0.6, and in specific embodiments it can be 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.55 or 1:0.6.

[0090] In the present invention, the time of the first hair removal treatment is preferably 90 to 150 minutes, and in specific embodiments may be 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes or 150 minutes.

[0091] In the present invention, the alcoholamine preferably includes one or more of triethanolamine, monoethanolamine and diethanolamine. In the present invention, the mass ratio of the pretreated animal skin to the alcoholamine is preferably 1:0.01-0.03, and in specific embodiments it can be 1:0.01, 1:0.015, 1:0.02, 1:0.025 or 1:0.03.

[0092] In the present invention, the time of the second hair removal treatment is preferably 10 to 20 minutes, and in specific embodiments it can be 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes or 20 minutes.

[0093] In the present invention, the alkaline agent preferably includes alkali metal hydroxide and / or alkali metal carbonate; the alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; the alkali metal carbonate preferably includes sodium carbonate and / or potassium carbonate. In the present invention, the mass ratio of the pretreated animal skin to the alkaline agent is preferably 1:0.035-0.045, and in specific embodiments it can be 1:0.035, 1:0.036, 1:0.037, 1:0.038, 1:0.039, 1:0.04, 1:0.041, 1:0.042, 1:0.043, 1:0.044 or 1:0.045.

[0094] In the present invention, the time of the third hair removal treatment is preferably 15 to 25 minutes, and in specific embodiments it can be 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes or 25 minutes.

[0095] In the present invention, the oxidant preferably includes one or more of hydrogen peroxide, sodium perborate and sodium percarbonate; the mass concentration of the hydrogen peroxide is preferably 1-30%, and in specific embodiments it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%, 25% or 30%. In the present invention, the mass ratio of the pretreated animal skin to the oxidant is preferably 1:0.06-0.08, and in specific embodiments it can be 1:0.061, 1:0.062, 1:0.063, 1:0.064, 1:0.065, 1:0.066, 1:0.067, 1:0.068, 1:0.069, 1:0.07, 1:0.071, 1:0.072, 1:0.073, 1:0.074, 1:0.075, 1:0.076, 1:0.077, 1:0.078, 1:0.079 or 1:0.08.

[0096] In the present invention, the duration of the fourth hair removal treatment is preferably 3 to 4 hours, and in specific embodiments may be 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes or 240 minutes.

[0097] In the present invention, the mass fraction of the dilute alkaline reagent aqueous solution is preferably 0.3-0.8%, and in specific embodiments it can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%; in the present invention, the alkaline reagent in the dilute alkaline reagent aqueous solution preferably includes alkali metal hydroxide and / or alkali metal carbonate. The alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; the alkali metal carbonate preferably includes sodium carbonate and / or potassium carbonate. The present invention has no special limitation on the amount of the dilute alkaline reagent aqueous solution, and it only needs to be able to immerse the animal skin after the fourth depilation treatment.

[0098] In the present invention, the fifth hair removal treatment is preferably performed for 4 to 6 hours, and in specific embodiments may be 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours.

[0099] In the present invention, the depilatory treatment preferably further comprises: washing the depilated animal skin with water to obtain the depilated animal skin. In the present invention, the number of times of washing with water is preferably 3 to 5 times, more preferably 4 times.

[0100] After obtaining the depilated animal skin, the present invention performs a deswelling treatment on the depilated animal skin to obtain the deswelling animal skin.

[0101] In the present invention, the deswelling treatment preferably includes: adding a first part of water and an inorganic salt to the dehaired animal skin for a first deswelling treatment, adding a second part of water, adjusting the pH value to 4-4.5, adding acid lipase for a second deswelling treatment, and adding acid protease for a third deswelling treatment; the deswelling treatment is preferably carried out in a rotating drum; the rotation speed of the rotating drum is preferably 5-12 r / min, and in specific embodiments it can be 5 r / min, 6 r / min, 7 r / min, 8 r / min, 9 r / min, 10 r / min, 11 r / min or 12 r / min.

[0102] In the present invention, the mass volume ratio of the pretreated animal skin to the first part of water is preferably 1kg:0.8-1L, and in specific embodiments it can be 1kg:0.8L, 1kg:0.85L, 1kg:0.9L, 1kg:0.95L or 1kg:1L.

[0103] In the present invention, the inorganic salt preferably includes one or more of sodium bisulfite, ammonium sulfate, ammonium chloride and ammonium acetate; the mass of the inorganic salt preferably accounts for 0.05-0.2% of the mass of the pretreated animal skin, and in specific embodiments it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2%.

[0104] In the present invention, the temperature of the first deswelling treatment is preferably 28-35°C, and in specific embodiments it can be 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C; the time of the first deswelling treatment is preferably 50-60min, and in specific embodiments it can be 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min or 60min; the endpoint pH value of the first deswelling treatment is preferably 8-8.5, and in specific embodiments the endpoint pH value can be 8, 8.1, 8.2, 8.3, 8.4 or 8.5.

[0105] In the present invention, the mass volume ratio of the pretreated animal skin to the second part of water is preferably 1kg:1-4L. In specific embodiments, it can be 1kg:1L, 1kg:1.1L, 1kg:1.2L, 1kg:1.5L, 1kg:2L, 1kg:2.5L, 1kg:3L, 1kg:3.5L or 1kg:4L.

[0106] In the present invention, the mass of the acid lipase preferably accounts for 0.1-0.3% of the mass of the pretreated animal skin, and in specific embodiments it can be 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, .27%, 0.28%, 0.29% or 0.3%; the enzyme activity of the acid lipase is preferably 50,000 to 60,000 units / g, and in specific embodiments may be 50,000 units / g, 51,000 units / g, 50,000 units / g, 53,000 units / g, 54,000 units / g, 55,000 units / g, 56,000 units / g, 57,000 units / g, 58,000 units / g, 59,000 units / g or 60,000 units / g;

[0107] In the present invention, the temperature of the second deswelling treatment is preferably 28-35°C, and in specific embodiments it can be 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C; the time of the second deswelling treatment is preferably 15-20 min, and in specific embodiments it can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0108] In the present invention, the mass of the acidic protease accounts for 0.1-0.3% of the mass of the pretreated animal skin, and in specific embodiments it can be 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29% or 0.3%; the enzyme activity of the acidic protease is preferably 80,000-100,000 units / g. In specific embodiments, the amount of the present invention may be 80,000 units / g, 81,000 units / g, 82,000 units / g, 83,000 units / g, 84,000 units / g, 85,000 units / g, 86,000 units / g, 87,000 units / g, 88,000 units / g, 89,000 units / g, 90,000 units / g, 91,000 units / g, 92,000 units / g, 93,000 units / g, 94,000 units / g, 95,000 units / g, 96,000 units / g, 97,000 units / g, 98,000 units / g, 99,000 units / g or 100,000 units / g.

[0109] In the present invention, the temperature of the third deswelling treatment is preferably 38-42°C, and in specific embodiments it can be 38°C, 39°C, 40°C, 41°C or 42°C; the time of the third deswelling treatment is preferably 2-5h, and in specific embodiments it can be 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h.

[0110] In the present invention, the deswelling treatment preferably further comprises: washing the deswelling treated animal skin with water to obtain the deswelling animal skin. In the present invention, the number of times of washing with water is preferably 3 to 5 times, more preferably 4 times.

[0111] After obtaining the de-swelling animal skin, the present invention performs acid swelling treatment on the de-swelling animal skin to obtain the acid-swollen animal skin.

[0112] In the present invention, the acid swelling treatment preferably includes: adding water to the de-swollen animal skin, adjusting the pH value to 2.8-3.0, stirring the acid swelling treatment and then allowing the acid swelling treatment to stand; the temperature of the acid swelling treatment is preferably room temperature, and the acid swelling treatment is preferably carried out in a rotating drum; the rotation speed of the rotating drum is preferably 5-12r / min, and in specific embodiments it can be 5r / min, 6r / min, 7r / min, 8r / min, 9r / min or 10r / min, 11r / min or 12r / min.

[0113] In the present invention, the mass volume ratio of the pretreated animal skin to water is preferably 1kg:1-2L. In specific embodiments, it can be 1kg:1L, 1kg:1.1L, 1kg:1.2L, 1kg:1.3L, 1kg:1.4L, 1kg:1.5L, 1kg:1.6L, 1kg:1.7L, 1kg:1.8L, 1kg:1.9L or 1kg:2L.

[0114] In the present invention, the acid used to adjust the pH value to 2.8-3.0 preferably includes formic acid solution and / or sulfuric acid solution; the mass concentration of the formic acid solution is preferably 0.05-0.08%, and in a specific embodiment it can be 0.05%, 0.06%, 0.07% or 0.08%; the mass concentration of the sulfuric acid solution is preferably 0.08-0.12%, and in a specific embodiment it can be 0.08%, 0.09%, 0.010%, 0.011% or 0.012%; when the acid is a mixed acid solution of formic acid solution and sulfuric acid solution, the volume ratio of formic acid solution to sulfuric acid solution in the mixed acid solution is preferably 1:2-3, more preferably 1:2.5.

[0115] In the present invention, the stirring acid swelling treatment time is preferably 30 to 60 minutes, and in specific embodiments it can be 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes.

[0116] In the present invention, the static acid swelling treatment time is preferably 6 to 8 hours, and in specific embodiments can be 6 hours, 6.5 hours, 7 hours, 7.5 hours or 8 hours.

[0117] In the present invention, the acid swelling treatment preferably further comprises: washing the animal skin after the acid swelling treatment to obtain the acid swollen animal skin. In the present invention, the water washing is preferably performed 3 to 5 times, more preferably 4 times.

[0118] After obtaining the acid-swollen animal skin, the present invention crushes the acid-swollen animal skin, adjusts the pH value to 7-7.4, separates the solid and liquid to obtain a solid component, washes the solid component with water and then dries it to obtain medical-grade tissue-like natural collagen.

[0119] In the present invention, the pulverizing preferably includes ball milling; the ball-to-material ratio of the ball milling is preferably 1:1-5, and in specific embodiments it can be 1kg:1-2L, 1kg:1-2L.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; the rotation speed of the ball milling is preferably 200-600r / min, and in specific embodiments it can be 200r / min, 300r / min, 400r / min, 500r / min or 600r / min; the time of the ball milling is preferably 1-3h, and in specific embodiments it can be 1h, 1.5h, 2h, 2.5h or 3h. In the present invention, the grinding bodies used in the ball mill preferably include large balls and small balls; the particle size of the large balls is preferably 12-13 mm; the particle size of the small balls is preferably 9-10 mm; the number ratio of the large balls to the small balls is preferably 1:1-3, and in specific embodiments it can be 1kg:1-2L, 1kg:1-2L.5, 1:2, 1:2.5 or 1:3.

[0120] After the ball milling, the present invention preferably further comprises centrifuging the ball milling system, removing the supernatant, collecting the slurry, and then adjusting the pH value of the slurry to 7 to 7.4. In the present invention, the rotation speed of the centrifugal separation is preferably 15000 to 18000 r / min, and in specific embodiments, it can be 15000 r / min, 16000 r / min, 17000 r / min or 18000 r / min; the time of the centrifugal separation is preferably 10 to 15 minutes, and in specific embodiments, it can be 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0121] In the present invention, the base used to adjust the pH value to 7-7.4 preferably includes one or more of hydroxides, carbonates and bicarbonates, and the hydroxide preferably includes one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide; the carbonate preferably includes sodium carbonate and / or potassium carbonate; the alkali metal bicarbonate preferably includes one or more of sodium bicarbonate, potassium bicarbonate and calcium bicarbonate; in a specific embodiment, the pH value can be 7, 7.1, 7.2, 7.3 or 7.4.

[0122] In the present invention, the solid-liquid separation preferably includes centrifugal separation, and the rotation speed of the centrifugal separation is preferably 15000-18000 r / min, and in specific embodiments it can be 15000 r / min, 16000 r / min, 17000 r / min or 18000 r / min; the time of the centrifugal separation is preferably 10-15 min, and in specific embodiments it can be 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0123] In the present invention, the drying preferably includes freeze drying. The present invention has no particular limitation on the conditions of freeze drying, and freeze drying is sufficient.

[0124] In the present invention, after drying, the method further preferably includes: sterilizing the dried natural collagen to obtain medical-grade tissue-like natural collagen. In the present invention, the sterilization is preferably γ-ray sterilization; the dose of the γ-ray is preferably 6 to 25 KGy / h60Co, and in specific embodiments, it can be 6 KGy / h60Co, 7 KGy / h60Co, 8 KGy / h60Co, 9 KGy / h60Co, 10 KGy / h60Co, 11 KGy / h60Co, 12 KGy / h60Co, 3 KGy / h60 Co、14KGy / h60Co、15KGy / h60Co、16KGy / h60Co、17KGy / h60Co、18KGy / h60Co、19KGy / h60C o, 20KGy / h60Co, 21KGy / h60Co, 22KGy / h60Co, 23KGy / h60Co, 24KGy / h60Co or 25KGy / h60Co.

[0125] The preparation method provided by the present invention has simple process, simple equipment, abundant raw materials, low cost, and easy large-scale industrial production. The medical-grade tissue-like natural collagen prepared by the present invention has not undergone any degradation process of natural collagen, and its basic composition is the existence form of natural collagen in the tissue-collagen fiber bundle, and also has the spatial structural characteristics of natural collagen in the tissue, so that it can mimic the natural tissue structure to a greater extent. The medical-grade tissue-like natural collagen prepared by the present invention has more excellent mechanical properties and excellent enzyme resistance / thermal stability and structural stability, low antigenicity and easier to preserve, excellent hemostatic performance, and its high strength and toughness can meet the body's diversified requirements for material properties, and is an ideal collagen biomedical material. The medical-grade tissue-like natural collagen prepared by the present invention is conducive to cell survival, proliferation and differentiation, and can significantly promote tissue regeneration and repair in wound and scar repair, and reduce the generation of scars.

[0126] The present invention also provides medical-grade tissue-like natural collagen prepared by the preparation method described in the above technical solution.

[0127] The present invention also provides the use of the medical-grade tissue-like natural collagen described in the above technical solution in tissue engineering, regenerative medicine materials or implantable medical devices. The medical-grade tissue-like natural collagen provided by the present invention has good biocompatibility, suitable biodegradability, good mechanical properties, excellent thermal stability and structural stability, low antigenicity and easy preservation, and can quickly and effectively stop bleeding and promote wound healing and repair, and is an ideal material for biomedical materials.

[0128] In order to further illustrate the present invention, the medical-grade tissue-like natural collagen provided by the present invention and its preparation method and application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0129] In the following examples, the unit ratio of mass fraction to volume fraction is kg:L.

[0130] Example 1

[0131] A) Pretreatment of cowhide: The cowhide was placed in a rotary drum and repeatedly washed to remove dirt on the cowhide, and then soaked in water for 60 minutes, and then mechanically cut to remove subcutaneous fat, surface muscles, blood vessels, fascia tissue and other subcutaneous tissues on the cowhide as much as possible, and then cut into small pieces of 5.0 mm × 5.0 mm, and fully washed with physiological saline at room temperature and soaked for 15 hours to obtain pretreated cowhide;

[0132] B) Degreasing of cowhide: 10 parts by weight of the above-mentioned pretreated cowhide were put into a rotary drum, 0.2 parts by weight of alkyl polyglycoside and 15 parts by volume of deionized water were added, and the cowhide was degreased at a constant temperature of 30° C. for 2 hours. After degreasing, the cowhide was repeatedly washed with warm water at 35° C. for 3 times to obtain degreased cowhide;

[0133] C) Dehairing of cowhide: the above-mentioned defatted cowhide is placed in water of equal weight, 4 parts by weight of fatty alcohol polyoxyethylene ether and 3 parts by weight of ferrous sulfate are added to the water, and after rotating in a drum for 1.5 hours, 0.1 parts by weight of triethanolamine is added, and after continuing to rotate for 10 minutes, 0.35 parts by weight of sodium hydroxide is added, and after rotating for 15 minutes, 0.6 parts by weight of hydrogen peroxide is added, and the cowhide is rotated for 3 hours. After the end, it is soaked in a sodium hydroxide solution with a mass fraction of 0.3% for 6 hours, and then repeatedly rinsed with ultrapure water for 3 times to obtain dehaired cowhide;

[0134] D) Deswelling treatment of cowhide: the dehaired cowhide is put into a drum, 8 parts by volume of ultrapure water is added, the temperature is controlled at 28°C, and 0.05% by weight of sodium bisulfite is added, and the drum is rotated for 60 minutes, and the end point pH value is controlled at about 8; after the rotation is completed, 10 parts by volume of deionized water and 3 parts by volume of formic acid are added to the same bath, and the pH value is controlled to be 4.5, and then 0.1% of acid lipase is added, and after rotating for 20 minutes, 0.3% of acid protease is added, and the reaction is carried out at a constant temperature of 40°C for 2 hours, and finally, it is repeatedly rinsed with ultrapure water for 3 times to obtain deswelling cowhide;

[0135] E) Acid swelling treatment of cowhide: 10 parts by volume of ultrapure water was added to the above de-swollen cowhide, and the pH value was adjusted to 2.8 with a mixed acid solution of 0.05-0.08wt% formic acid solution and 0.08-0.12wt% sulfuric acid solution (volume ratio = 1:2), stirred for 0.5h, and rinsed repeatedly with ultrapure water for 3 times after standing for 6h to obtain acid-swollen cowhide;

[0136] F) Preparation of tissue-like natural collagen: Take the above acid-swollen cowhide, put it and the material ball into a ball milling jar, make the ball-to-material ratio be 1:1, ball mill for 180min at a speed of 200r / min, centrifuge the slurry at 15000r / min for 15min after grinding, remove the supernatant, collect the slurry, adjust the pH of the slurry to 7.0 with sodium hydroxide, centrifuge at 15000r / min for 15min, collect the precipitate, wash the precipitate with ultrapure water for 3 times, finally freeze-dry, sterilize with γ-rays produced by 60Co at a dose of 6KGy / h, shape and package, and obtain medical-grade tissue-like natural collagen. Among them, the grinding bodies used in the ball mill are large balls with a particle size of 12-13mm and small balls with a particle size of 9-10mm, and the number ratio of large balls to small balls is 1:3. The drum speed in this embodiment is 12r / min.

[0137] Example 2

[0138] A) Pretreatment of pigskin: The pigskin was placed in a rotary drum for repeated washing to remove dirt on the pigskin, and then soaked in water for 90 minutes. The pigskin was then subjected to mechanical meat cutting to remove subcutaneous fat, surface muscles, blood vessels, fascia tissue and other subcutaneous tissues on the pigskin as much as possible. The pigskin was then cut into small pieces of 8.0 mm × 8.0 mm, and fully washed with physiological saline at room temperature and soaked for 13 hours to obtain pretreated pigskin;

[0139] B) Pigskin degreasing treatment: 10 parts by weight of the above-mentioned pretreated pigskin is put into a rotary drum, 0.3 parts by weight of alkyl polyglycoside and 20 parts by volume of deionized water are added, and the pigskin is degreased at a constant temperature of 35° C. for 3 hours. After degreasing, the pigskin is repeatedly washed with warm water at 38° C. for 4 times to obtain defatted pigskin;

[0140] C) Pigskin depilation: The above-mentioned defatted pigskin is placed in water of equal weight, 5 parts by weight of fatty alcohol polyoxyethylene ether and 4 parts by weight of ferrous sulfate are added to the water, and after rotating in a drum for 2 hours, 0.2 parts by weight of triethanolamine is added, and after continuing to rotate for 15 minutes, 0.4 parts by weight of sodium hydroxide is added, and after rotating for 20 minutes, 0.7 parts by weight of hydrogen peroxide is added, and the skin is rotated for 3.5 hours. After the end, it is soaked in a sodium hydroxide solution with a mass fraction of 0.5% for 5 hours, and then rinsed repeatedly with ultrapure water for 4 times to obtain a depilated pigskin;

[0141] D) Pigskin deswelling treatment: The dehaired pigskin is put into a drum, 9 parts by volume of ultrapure water is added, the temperature is controlled at 30° C., and sodium bisulfite with a tare weight of 0.12% is added, and the drum is rotated for 55 minutes, and the end point pH value is controlled at about 8.3; after the rotation is completed, 10 parts by volume of deionized water and 3.5 parts by volume of formic acid are added to the same bath, the pH value is controlled to be 4.2, and then 0.2% of acid lipase is added, and after rotating for 20 minutes, 0.2% of acid protease is added, and the reaction is carried out at a constant temperature of 40° C. for 3 hours. Finally, the deswelling pigskin is repeatedly rinsed 4 times with ultrapure water to obtain a deswelling pigskin;

[0142] E) Acid swelling treatment of pig skin: 15 parts by volume of ultrapure water was further added to the de-swollen pig skin, and the pH value was adjusted to 2.9 with a mixed acid solution of 0.05-0.08wt% formic acid solution and 0.08-0.12wt% sulfuric acid solution (volume ratio = 1:2), stirred for 0.8h, and rinsed repeatedly with ultrapure water for 4 times after standing for 7h to obtain acid swollen pig skin;

[0143] F) Preparation of tissue-like natural collagen: Take the above acid-swollen pigskin, put it and the material ball into a ball mill, make the ball-to-material ratio 1:3, ball mill for 120min at a speed of 400r / min, centrifuge the slurry at 16000r / min for 13min after grinding, remove the supernatant, collect the slurry, adjust the pH of the slurry to 7.2 with sodium carbonate, centrifuge at 16000r / min for 13min, collect the precipitate, wash the precipitate with ultrapure water 4 times, finally freeze-dry, sterilize with γ-rays produced by 18KGy / h60Co, shape and package, and obtain medical-grade tissue-like natural collagen. Among them, the grinding bodies used in the ball mill are large balls with a particle size of 12-13mm and small balls with a particle size of 9-10mm, and the number ratio of large balls to small balls is 1:2. The drum speed in this embodiment is 10r / min.

[0144] Example 3

[0145] A) Sheepskin pretreatment: sheepskin was placed in a rotary drum for repeated washing to remove dirt on the sheepskin, and then soaked in water for 120 min after washing, and then mechanically cut to remove subcutaneous fat, surface muscles, blood vessels, fascia tissue and other subcutaneous tissues on the sheepskin as much as possible, and then cut into small pieces of 10.0 mm × 10.0 mm, and fully washed with physiological saline at room temperature and soaked for 10 h to obtain pretreated sheepskin;

[0146] B) Sheepskin degreasing: 10 parts by weight of the pretreated sheepskin are put into a rotary drum, 0.4 parts by weight of alkyl polyglycoside and 25 parts by volume of deionized water are added, and the sheepskin is degreased at a constant temperature of 37° C. for 5 hours. After degreasing, the sheepskin is repeatedly washed with warm water at 40° C. for 5 times;

[0147] C) Sheepskin depilation: The above-mentioned defatted sheepskin is placed in water of equal weight, 6 parts by weight of fatty alcohol polyoxyethylene ether and 5 parts by weight of ferrous sulfate are added to the water, and after rotating in a drum for 2.5 hours, 0.3 parts by weight of triethanolamine is added, and after continuing to rotate for 20 minutes, 0.45 parts by weight of sodium hydroxide is added, and after rotating for 25 minutes, 0.8 parts by weight of hydrogen peroxide is added, and the sheepskin is rotated for 4 hours. After the end, it is soaked in a sodium hydroxide solution with a mass fraction of 0.8% for 4 hours, and then rinsed repeatedly with ultrapure water for 5 times;

[0148] D) Sheepskin swelling removal treatment: The dehaired sheepskin is put into a drum, 10 parts by volume of ultrapure water is added, the temperature is controlled at 35° C., and 0.2% sodium bisulfite by weight is added, and the drum is rotated for 50 minutes, and the end point pH value is controlled at about 8.5; after the rotation is completed, 10 parts by volume of deionized water and 4 parts by volume of formic acid are added to the same bath, and the pH value is controlled to be 4, and then 0.3% of acid lipase is added, and after rotating for 20 minutes, 0.1% of acid protease is added, and the reaction is carried out at a constant temperature of 40° C. for 5 hours, and finally, it is repeatedly rinsed with ultrapure water for 5 times;

[0149] E) Acid swelling treatment of sheepskin: 20 parts by volume of ultrapure water were added to the above-mentioned de-swollen sheepskin, and the pH value was adjusted to 3.0 with a mixed acid solution of 0.05-0.08wt% formic acid solution and 0.08-0.12wt% sulfuric acid solution (volume ratio = 1:2), stirred for 1 hour, and rinsed repeatedly with ultrapure water for 5 times after standing for 8 hours;

[0150] F) Preparation of tissue-like natural collagen: Take the above-mentioned acid-swollen sheepskin, put it and the material ball into a ball milling jar, make the ball-to-material ratio be 1:5, ball mill for 60 minutes at a speed of 600r / min, centrifuge the slurry at 18000r / min for 10 minutes after grinding, remove the supernatant, collect the slurry, adjust the pH of the slurry to 7.4 with sodium bicarbonate, centrifuge at 18000r / min for 10 minutes, collect the precipitate, wash the precipitate with ultrapure water for 5 times, finally freeze-dry, sterilize with γ-rays produced by 25KGy / h60Co, shape and package, and obtain medical-grade tissue-like natural collagen. Among them, the grinding bodies used in the ball mill are large balls with a particle size of 12-13mm and small balls with a particle size of 9-10mm, and the number ratio of large balls to small balls is 1:1. The speed of the drum in this embodiment is 7r / min.

[0151] Figure 1 This is an atomic force microscope (AFM) detection image of the medical-grade tissue-like natural collagen prepared in Example 1. As can be seen from Figure 1, the prepared tissue-like natural collagen presents the typical D-periodic light and dark alternating horizontal stripe structure of natural collagen, indicating that it completely retains the original aggregation structure of natural collagen.

[0152] Comparative Example 1

[0153] Antigen-free collagen aggregates prepared in Example 2 of Chinese Patent CN104107456A.

[0154] Comparative Example 1 uses acetone and supercritical fluid for degreasing. Acetone has certain toxicity, and the industrialization of supercritical fluid devices is relatively complicated. Under the condition of achieving the same degreasing effect, the present invention uses alkyl polyglycosides with better biocompatibility, and the equipment also uses conventional leather equipment drums, which are more suitable for industrial applications. The preparation method provided by the present invention adds a depilatory process relative to Comparative Example 1. This process can further remove non-collagen components in the skin such as miscellaneous proteins and fiber stroma while removing hair, further loosen the collagen fibers in the skin, facilitate the subsequent extraction of natural collagen, and improve the extraction rate of natural collagen. Comparative Example 1 uses a homogenizer beating process, and inevitably a large number of small particles of leather pieces are retained because the beating process cannot ensure complete homogenization, affecting subsequent applications; while the present invention uses a ball milling process that is more in line with industrial production. The preparation cycle under this process is shorter, the extraction rate of natural collagen is higher, and the ball milling process used is more uniform in sample preparation, and the quality is more controllable.

[0155] Test Example 1

[0156] Performance testing of collagen materials (medical-grade tissue-like natural collagen prepared in Example 2, antigen-free collagen aggregates prepared in Comparative Example 1, and commercially available molecular-grade collagen).

[0157] (1) The results of collagen extraction rate and impurity protein content of collagen materials are shown in Table 1.

[0158] Table 1 Collagen extraction rate and impurity protein content of collagen materials

[0159] collagen Collagen extraction rate Miscellaneous protein content Comparative Example 1 75％ 0.8wt% Example 2 92％ 0.4wt%

[0160] It can be seen from Table 1 that, compared with Comparative Example 1, the collagen extraction rate of the medical-grade tissue-like natural collagen prepared by the present invention is significantly improved, and the impurity protein content is significantly reduced.

[0161] (2) The weight loss of collagen materials at different temperature ranges is shown in Table 2.

[0162] Table 2 Weight loss of collagen materials at different temperature ranges

[0163]

[0164]

[0165] It can be seen from Table 2 that, compared with Comparative Example 1 and collagen, the medical-grade tissue-like natural collagen prepared by the present invention has a lower weight loss rate in the temperature range of 50 to 800° C., and has better thermal stability and structural stability.

[0166] (3) 30-day degradation rate test method: Degradation in collagenase solution, the specific operation is as follows: bacterial type I collagenase is dissolved in PBS buffer to prepare a type I collagenase degradation solution (1U / mL) with a concentration of 2mL / mg. The samples are freeze-dried and immersed in the degradation solution for 30 days. After that, the degradation experiment is carried out in a 37°C constant temperature biochemical incubator.

[0167] Tensile strength test method: Pour the collagen sample into a polytetrafluoroethylene mold to cast it into a film, and then use a universal tensile testing machine to test the tensile strength of the sample.

[0168] Hemostasis time test method: rabbit ear central artery hemostasis model.

[0169] The hemostatic effect, degradation rate and tensile strength of collagen materials are shown in Table 3.

[0170] Table 3 Hemostasis, degradation and tensile strength results of collagen materials

[0171] sample Bleeding time 30-day degradation rate Tensile Strength Example 2 68s 68％ 3.2MPa Comparative Example 1 116s 89％ 2.4MPa collagen 182s 100％ 0.9MPa

[0172] It can be seen from Table 3 that, compared with Comparative Example 1 and collagen, the medical-grade tissue-like natural collagen prepared by the present invention has a shorter hemostatic time and a better hemostatic effect; a lower 30-day degradation rate and a slower degradation speed; and a higher tensile strength.

[0173] (4) MTT assay

[0174] Experimental method: Use culture medium containing 10% fibroblasts to prepare a single cell suspension, inoculate 1000-10000 cells per well into a 96-well plate, and the volume of each well is 200 μL. Under standard culture conditions, maintain cell culture for 3-5 days. After the culture period, add 10 μL of MTT solution to each well and continue incubation for 4 hours. Terminate the culture and carefully discard the culture supernatant in the well. For suspended cells, centrifuge and then discard the culture supernatant in the well. After that, add 100 μL of dimethyl sulfoxide (DMSO) to each well and shake for 10 minutes to fully dissolve the crystals. Finally, select a wavelength of 490 nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, and record the results. Draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis to evaluate the growth of cells.

[0175] Figure 2 From the MTT test results of collagen, the antigen-free collagen aggregates prepared in Comparative Example 1, and the medical-grade tissue-like natural collagen prepared in Example 2, it can be seen that the medical-grade tissue-like natural collagen prepared by the present invention provides a more suitable growth environment for cells, shows significant advantages in promoting the proliferation and growth of fibroblasts, and provides a more ideal growth microenvironment for cells.

[0176] (5) 24h scratch test

[0177] Experimental method: Use a marker pen to draw horizontal lines on the back of a six-well plate with a ruler as a reference for subsequent experiments. Inoculate 3×10 5 cells, with a total volume of 2 mL per well; after culturing the cells for 24 hours, use a 200 μL pipette tip to scratch the cell layer along the pre-marked line to form a "wound"; after the scratching is completed, the culture supernatant is aspirated, and the cells are washed 2 to 3 times with PBS to remove the scratched cells; the old culture medium is discarded, and the medium containing drugs or serum-free medium is added according to the experimental grouping; the cells are cultured in a 37°C, 5% CO2 incubator. The cells are taken out at predetermined time points such as 0h and 24h, and the changes in the width of the scratches at the same position are observed and recorded under a microscope, and photos are taken to observe the changes in cell migration.

[0178] Figure 3 This is the 24h scratch result of collagen. Figure 4 This is the 24h scratch result of the antigen-free collagen aggregate prepared in Comparative Example 1. Figure 5 This is the 24h scratch result of the medical-grade natural tissue collagen prepared in Example 2. Figure 3 to Figure 5 It can be seen that compared with collagen and the antigen-free collagen aggregate prepared in Comparative Example 1, the medical-grade tissue-like natural collagen prepared by the present invention can better promote cell growth and proliferation, and is more conducive to wound tissue regeneration and reducing scar formation.

[0179] (6) Tissue HE staining

[0180] Figure 6 This is an HE-stained section of animal skin tissue of the medical-grade natural tissue collagen prepared in Example 2 (×100). As can be seen from Figure 6, the collagen fiber bundles in the medical-grade natural tissue collagen prepared by the present invention are clean and free of impurities, the fibers remain intact, and the product is confirmed to be qualified.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing medical grade tissue-like natural collagen, comprising the following steps: removing subcutaneous tissue of the animal skin and soaking it in physiological saline to obtain pretreated animal skin; Degreasing the pretreated animal skin to obtain defatted animal skin; Dehairing the defatted animal skin to obtain a dehaired animal skin; performing a deswelling treatment on the depilated animal skin to obtain a deswelling animal skin; subjecting the de-swollen animal skin to an acid swelling treatment to obtain an acid swollen animal skin; The acid-swollen animal skin is crushed, the pH value is adjusted to 7-7.4, the solid-liquid separation is carried out to obtain a solid component, the solid component is washed with water and then dried to obtain medical-grade tissue-like natural collagen.

2. The preparation method according to claim 1, characterized in that: The degreasing treatment comprises: adding a degreasing agent and water to the pretreated animal skin to perform degreasing treatment; The mass ratio of the pretreated animal skin to the degreasing agent is 1:0.01-0.04; The degreasing agent includes one or more of alkyl polyglycosides, peregrin, washing powder, sodium lauryl sulfate and fatty alcohol polyoxyethylene ether; The mass volume ratio of the pretreated animal skin to water is 1kg:1.5-3L; The degreasing treatment is performed at a temperature of 30 to 37°C for 2 to 5 hours; The degreasing treatment further includes water washing.

3. The preparation method according to claim 1, characterized in that: The depilatory treatment comprises: adding the defatted animal skin, water, polyoxyethylene ether and ferrous salt to perform a first depilatory treatment, adding alcohol amine to perform a second depilatory treatment, adding an alkaline agent to perform a third depilatory treatment, adding an oxidant to perform a fourth depilatory treatment, and adding a dilute alkaline agent aqueous solution to perform a fifth depilatory treatment; The mass volume ratio of the defatted animal skin and water is 1kg:1-2L; The mass ratio of the pretreated animal skin to the polyoxyethylene ether is 1:0.3-0.6; The mass ratio of the pretreated animal skin to the ferrous salt is 1:0.3-0.6; The first hair removal treatment lasts for 90 to 150 minutes; The mass ratio of the pretreated animal skin to the alcohol amine is 1:0.01-0.03; The second hair removal treatment lasts for 10 to 20 minutes; The mass ratio of the pretreated animal skin to the alkaline reagent is 1:0.035-0.045; The third hair removal treatment lasts for 15 to 25 minutes; The mass ratio of the pretreated animal skin to the oxidant is 1:0.06-0.08; The fourth hair removal treatment lasts for 3 to 4 hours; The mass fraction of the dilute alkaline reagent aqueous solution is 0.3-0.8%; the time of the fifth depilatory treatment is 4-6 hours; The depilatory treatment further comprises washing with water.

4. The preparation method according to claim 3, characterized in that: The polyoxyethylene ether includes fatty alcohol polyoxyethylene ether and / or nonylphenol polyoxyethylene ether; The ferrous salt includes ferrous sulfate and / or ammonium ferrous sulfate; The alcohol amine includes one or more of triethanolamine, monoethanolamine and diethanolamine; The alkaline agent includes alkali metal hydroxide and / or alkali metal carbonate; The oxidant includes one or more of hydrogen peroxide, sodium perborate and sodium percarbonate; The alkaline agent in the dilute alkaline agent aqueous solution includes alkali metal hydroxide and / or alkali metal carbonate.

5. The preparation method according to claim 1, characterized in that: The deswelling treatment comprises: adding a first portion of water and an inorganic salt to the dehaired animal skin for a first deswelling treatment, adding a second portion of water, adjusting the pH value to 4 to 4.5, adding acid lipase for a second deswelling treatment, and adding acid protease for a third deswelling treatment; The mass volume ratio of the pretreated animal skin to the first portion of water is 1 kg: 0.8 to 1 L; The inorganic salt includes one or more of sodium bisulfite, ammonium sulfate, ammonium chloride and ammonium acetate; the mass of the inorganic salt accounts for 0.05-0.2% of the mass of the pretreated animal skin; The temperature of the first deswelling treatment is 28-35°C, the time is 50-60 minutes, and the end point pH value is 8-8.5; The mass volume ratio of the pretreated animal skin to the second part of water is 1 kg: 1 to 4 L; The mass of the acid lipase accounts for 0.1-0.3% of the mass of the pretreated animal skin; the enzyme activity of the acid lipase is 50,000-60,000 units / g; The second deswelling treatment is performed at a temperature of 28 to 35°C for 15 to 20 minutes; The mass of the acidic protease accounts for 0.1-0.3% of the mass of the pretreated animal skin; the enzyme activity of the acidic protease is 80,000-100,000 units / g; The temperature of the third deswelling treatment is 38-42°C and the time is 2-5 hours; The deswelling treatment further comprises water washing.

6. The preparation method according to claim 1, characterized in that: The acid swelling treatment comprises: adding water to the de-swelled animal skin, adjusting the pH value to 2.8-3.0, stirring for acid swelling treatment and then standing for acid swelling treatment; The mass volume ratio of the pretreated animal skin to water is 1 kg: 1 to 2 L; The stirring acid swelling treatment time is 30 to 60 minutes; The time of the static acid swelling treatment is 6 to 8 hours; The acid swelling treatment further includes water washing.

7. The preparation method according to claim 1, characterized in that: The pulverization includes ball milling, wherein the ball-to-material ratio of the ball milling is 1:1-5, the rotation speed is 200-600r / min, and the time is 1-3h; The drying comprises freeze drying; The drying process also includes disinfection and sterilization.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The animal skin includes one or more of the skins of pigs, cows and sheep.

9. Medical grade tissue-like natural collagen obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the medical-grade tissue-like natural collagen according to claim 9 in tissue engineering, regenerative medicine materials or implantable medical devices.

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