A medical grade tissue-like natural collagen and its preparation method and application

Abstract

The application provides a medical grade tissue-like natural collagen and a preparation method and application thereof, and relates to biomedical materials. The application provides a preparation method of the medical grade tissue-like natural collagen, which comprises the following steps: removing subcutaneous tissue of animal skin, soaking in physiological saline, sequentially performing defatting treatment, hair removal treatment, swelling removal treatment, acid swelling treatment, ball milling, adjusting pH value to 7-7.4, performing solid-liquid separation, and drying the obtained solid component to obtain the medical grade tissue-like natural collagen. The medical grade tissue-like natural collagen prepared by the application has good biocompatibility, suitable biodegradability, good mechanical properties, excellent thermal stability and structural stability, low antigenicity and easy preservation, can quickly and effectively stop bleeding, promote wound healing and repair, and is an ideal material of biomedical materials.

Description

TECHNICAL FIELD

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

[0002] Collagen is the most abundant and widely distributed structural protein in animals, widely exists in skin, Achilles tendon, cartilage, cornea and various connective tissues. Collagen makes the extracellular matrix have good mechanical properties and structural integrity, has the functions of supporting organs and protecting the body, and plays a key role in maintaining the structure and physiological function of the body. The structure and morphology of collagen in living organisms have always been a research hotspot in the field of collagen. It has been confirmed that the existence of natural collagen in tissues is not at the level of collagen molecules with triple helix structure on the market, nor is it simply a single collagen fiber. The complex physiological aggregation process in living organisms makes it have typical multi-level structure characteristics. The collagen molecules with triple helix structure synthesized by amino acids are arranged in bundles in a staggered quarter way, and are cross-linked to form stable collagen microfibers through covalent bonds, and then are orderly arranged in a ladder form to aggregate into fibrils with a diameter of 50-200nm and a length of 280nm to several microns, and then a bundle of microfibers is formed. A bundle of microfibers can form a fibril. The fibrils are further wound together to form a collagen fiber bundle. The collagen fiber bundle forms the basic fibrous structure of the three-dimensional network of living organisms through mutual division and interpenetration. The basic form of natural collagen in tissues is collagen fiber bundle.

[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 acid enzyme combination method. However, since the method involves the degradation process of natural collagen, it greatly destroys the aggregate structure of natural collagen, resulting in application limitations such as insufficient mechanical properties and low thermal stability of the prepared molecular level collagen materials. SUMMARY

[0004] Therefore, the present application aims to provide a medical grade tissue-like natural collagen and a preparation method and application thereof. The medical grade tissue-like natural collagen prepared by the preparation method provided by the present application has excellent mechanical properties, thermal stability and hemostatic performance, and slow degradation, which can significantly promote the regeneration and repair of tissues and reduce the generation of scars.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a preparation method of a medical grade tissue-like natural collagen, comprising the following steps:

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

[0008] subjecting the pretreated animal skin to degreasing treatment to obtain degreased animal skin;

[0009] subjecting the degreased animal skin to unhairing treatment to obtain unhairing animal skin;

[0010] subjecting the unhairing animal skin to swelling removal treatment to obtain swelling removal animal skin;

[0011] subjecting the swelling removal animal skin to acid swelling treatment to obtain acid swelling animal skin;

[0012] subjecting the acid swelling animal skin to crushing, adjusting pH value to 7-7.4, solid-liquid separation, obtaining solid component, and subjecting the solid component to water washing and drying to obtain medical grade tissue-like natural collagen.

[0013] Preferably, the degreasing treatment comprises adding degreasing agent and water to the pretreated animal skin for 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 comprises one or more of alkyl polyglycoside, perpass, washing powder, sodium dodecyl sulfate and fatty alcohol polyoxyethylene ether.

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

[0017] The temperature of the degreasing treatment is 30-37℃, and the time is 2-5 h.

[0018] The degreasing treatment further comprises water washing.

[0019] Preferably, the unhairing treatment comprises adding the degreased animal skin, water, polyoxyethylene ether and ferrous salt for first unhairing treatment, adding alcohol amine for second unhairing treatment, adding alkaline reagent for third unhairing treatment, adding oxidizing agent for fourth unhairing treatment, and adding dilute alkaline aqueous solution for fifth unhairing treatment.

[0020] The mass-volume ratio of the degreased animal skin to water is 1 kg:1-2 L.

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

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

[0023] The first unhairing treatment lasts for 90-150 minutes;

[0024] The mass ratio of the pre-treated animal hide to the alcohol amine is 1:0.01-0.03;

[0025] The second unhairing treatment lasts for 10-20 minutes;

[0026] The mass ratio of the pre-treated animal hide to the alkaline agent is 1:0.035-0.045;

[0027] The third unhairing treatment lasts for 15-25 minutes;

[0028] The mass ratio of the pre-treated animal hide to the oxidizing agent is 1:0.06-0.08;

[0029] The fourth unhairing treatment lasts for 3-4 hours;

[0030] The mass fraction of the dilute alkaline agent aqueous solution is 0.3-0.8%; the fifth unhairing treatment lasts for 4-6 hours;

[0031] The unhairing treatment further comprises water washing.

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

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

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

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

[0036] The oxidizing agent comprises one or more of hydrogen peroxide, sodium perborate and sodium percarbonate;

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

[0038] Preferably, the de-swelling treatment comprises: adding a first portion of water and an inorganic salt to the unhairing animal hide to perform a first de-swelling treatment, adding a second portion of water, adjusting the pH value to 4-4.5, adding an acid lipase to perform a second de-swelling treatment, and adding an acid protease to perform a third de-swelling treatment;

[0039] The mass-volume ratio of the pre-treated animal hide to the first portion of water is 1 kg: 0.8-1 L;

[0040] The inorganic salt comprises 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 hide;

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

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

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

[0044] The temperature of the second deswelling treatment is 28-35℃, and the time is 15-20 min;

[0045] The mass of the acid protease accounts for 0.1-0.3% of the mass of the pretreated animal hide; the enzyme activity of the acid protease is 80-100 thousand units / g;

[0046] The temperature of the third deswelling treatment is 38-42℃, and the time is 2-5 h;

[0047] The deswelling treatment further comprises water washing.

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

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

[0050] The stirring time of the acid swelling treatment is 30-60 min;

[0051] The standing time of the acid swelling treatment is 6-8 h;

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

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

[0054] The drying comprises freeze-drying;

[0055] The drying further comprises sterilization.

[0056] Preferably, the animal hide comprises animal hide of one or more of pig, cow and sheep.

[0057] The application further provides the medical grade tissue-like natural collagen prepared by the preparation method.

[0058] The application further provides application of the medical grade tissue-like natural collagen in tissue engineering, regenerative medicine materials or implantable medical devices.

[0059] The application provides a preparation method of medical grade tissue-like natural collagen, comprising the following steps: removing subcutaneous tissue of animal skin, soaking in physiological saline, sequentially performing degreasing treatment, hair removal treatment, swelling removal 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 drying to obtain the medical grade tissue-like natural collagen. The medical grade tissue-like natural collagen prepared by the application has more excellent mechanical properties, excellent enzyme / thermal stability and structural stability, low antigenicity and is easier to store, and has excellent hemostatic performance, and the high strength and toughness of the medical grade tissue-like natural collagen can meet the diversified requirements of the body on material performance, so that the medical grade tissue-like natural collagen is an ideal collagen biomaterial. The medical grade tissue-like natural collagen prepared by the application is beneficial to the survival, proliferation and differentiation of cells, can significantly promote the regeneration and repair of tissues in wound and scar repair, and reduce the generation of scars.

[0060] Moreover, the preparation method provided by the application has simple process, simple required equipment, rich raw material sources, low cost and easy scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 An atomic force microscope (AFM) detection graph of the medical grade tissue-like natural collagen prepared for Example 1;

[0062] Figure 2 MTT experiment results of collagen, the antigen-free collagen aggregate prepared for Comparative Example 1 and the medical grade tissue-like natural collagen prepared for Example 2;

[0063] Figure 3 A 24h scratch result graph of collagen;

[0064] Figure 4 A 24h scratch result graph of the antigen-free collagen aggregate prepared for Comparative Example 1;

[0065] Figure 5 A 24h scratch result graph of the medical grade tissue-like natural collagen prepared for Example 2;

[0066] Figure 6 HE staining section view of animal skin tissue of medical grade tissue-like natural collagen prepared in Example 2 (x100). DETAILED DESCRIPTION

[0067] The application provides a preparation method of medical grade tissue-like natural collagen, comprising the following steps:

[0068] The subcutaneous tissue of the animal skin is removed, and the animal skin is soaked in physiological saline to obtain pretreated animal skin.

[0069] The pretreated animal skin is subjected to degreasing treatment to obtain degreased animal skin.

[0070] The degreased animal skin is subjected to hair removal treatment to obtain hair-removed animal skin.

[0071] The hair-removed animal skin is subjected to swelling removal treatment to obtain swelling-removed animal skin.

[0072] The swelling-removed animal skin is subjected to acid swelling treatment to obtain acid-swollen animal skin.

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

[0074] Unless otherwise specified, the materials and equipment used in the application are commercially available in the art.

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

[0076] In the application, the animal skin preferably comprises animal skin of one or more of pigs, cattle and sheep; and the animal skin is preferably fresh and traceable animal skin.

[0077] Preferably, the animal skin is first cleaned and soaked in water, and then the subcutaneous tissue of the animal skin is removed. In the application, the purpose of the cleaning is to remove dirt on the animal skin. In the application, the temperature of the water soaking is preferably room temperature, and the time of the water soaking is preferably 60-120 min, which can be 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min in specific embodiments. In the application, the method of removing the subcutaneous tissue of the animal skin preferably comprises mechanical meat cutting treatment; and the subcutaneous tissue comprises subcutaneous fat, surface muscle, blood vessels and fascial tissue.

[0078] Preferably, the animal hide from which the subcutaneous tissue has been removed is cut into pieces, washed with physiological saline, and then soaked in physiological saline. In the present application, the size of the animal pieces obtained by cutting is preferably 5-10 mm x 5-10 mm. In the present application, the temperature of the physiological saline soaking is preferably room temperature, and the time of the physiological saline soaking is preferably 10-18 h, which in specific embodiments can be 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, or 18 h.

[0079] After obtaining the pretreated animal hide, the pretreated animal hide is subjected to a degreasing treatment in the present application to obtain a degreased animal hide.

[0080] In the present application, the degreasing treatment preferably comprises adding a degreasing agent and water to the pretreated animal hide and performing a degreasing treatment.

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

[0082] In the present application, the degreasing agent preferably comprises one or more of alkyl polyglycoside, peregalyn, laundry detergent, sodium dodecyl sulfate, and fatty alcohol polyoxyethylene ether. In the present application, the mass ratio of the pretreated animal hide to the degreasing agent is preferably 1: 0.01-0.04, which in specific embodiments 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 application, the temperature of the degreasing treatment is preferably 30-37℃, which in specific embodiments can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, or 37℃; the time of the degreasing treatment is preferably 2-5 h, which in specific embodiments can be 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, or 5 h; the degreasing treatment is preferably performed in a rotating drum; and the rotating speed of the rotating drum is preferably 5-12 r / min, which in specific embodiments 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.

[0084] In the present application, the defatting treatment is preferably followed by water washing of the defatted animal hide to obtain a defatted animal hide. In the present application, the number of water washing is preferably 3-5 times, more preferably 4 times.

[0085] After obtaining the defatted animal hide, the defatted animal hide is subjected to unhairing treatment to obtain an unhairing animal hide.

[0086] In the present application, the unhairing treatment preferably comprises: adding the defatted animal hide, water, polyoxyethylene ether and ferrous salt for first unhairing treatment, adding alcohol amine for second unhairing treatment, adding alkaline reagent for third unhairing treatment, adding oxidizing agent for fourth unhairing treatment, and adding dilute alkaline reagent aqueous solution for fifth unhairing treatment. In the present application, the temperature of the unhairing treatment is preferably room temperature, and the unhairing treatment is preferably carried out in a rotating drum; the rotating speed of the rotating drum is preferably 5-12 r / min, which in specific embodiments 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 application, the mass-volume ratio of the defatted animal hide and water is preferably 1 kg: 1-2 L, which in specific embodiments can be 1 kg: 1 L, 1 kg: 1.1 L, 1 kg: 1.2 L, 1 kg: 1.3 L, 1 kg: 1.4 L, 1 kg: 1.5 L, 1 kg: 1.6 L, 1 kg: 1.7 L, 1 kg: 1.8 L, 1 kg: 1.9 L or 1 kg: 2 L.

[0088] In the present application, the polyoxyethylene ether preferably comprises fatty alcohol polyoxyethylene ether and / or nonylphenol polyoxyethylene ether. In the present application, the mass ratio of the pretreated animal hide to polyoxyethylene ether is preferably 1: 0.3-0.6, which in specific embodiments 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 application, the ferrous salt preferably comprises ferrous sulfate and / or ferrous ammonium sulfate. In the present application, the mass ratio of the pretreated animal hide to ferrous salt is preferably 1: 0.3-0.6, which in specific embodiments 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 application, the time of the first unhairing treatment is preferably 90-150 min, which in specific embodiments can be 90 min, 100 min, 110 min, 120 min, 130 min, 140 min or 150 min.

[0091] In the present application, the alcohol amine preferably comprises one or more of triethanolamine, monoethanolamine and diethanolamine. In the present application, the mass ratio of the pretreated animal hide to the alcohol amine is preferably 1:0.01-0.03, and in specific embodiments can be 1:0.01, 1:0.015, 1:0.02, 1:0.025 or 1:0.03.

[0092] In the present application, the time of the second depilation treatment is preferably 10-20 min, and in specific embodiments can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0093] In the present application, the alkaline agent preferably comprises an alkali metal hydroxide and / or an alkali metal carbonate; the alkali metal hydroxide preferably comprises sodium hydroxide and / or potassium hydroxide; and the alkali metal carbonate preferably comprises sodium carbonate and / or potassium carbonate. In the present application, the mass ratio of the pretreated animal hide to the alkaline agent is preferably 1:0.035-0.045, and in specific embodiments 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 application, the time of the third depilation treatment is preferably 15-25 min, and in specific embodiments can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min or 25 min.

[0095] In the present application, the oxidizing agent preferably comprises 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 can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%, 25% or 30%. In the present application, the mass ratio of the pretreated animal hide to the oxidizing agent is preferably 1:0.06-0.08, and in specific embodiments 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 application, the time of the fourth depilation treatment is preferably 3-4 hours, and in specific embodiments can be 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes or 240 minutes.

[0097] In the present application, the mass fraction of the dilute alkaline reagent aqueous solution is preferably 0.3-0.8%, and in specific embodiments can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%; in the present application, the alkaline reagent in the dilute alkaline reagent aqueous solution preferably includes an alkali metal hydroxide and / or an alkali metal carbonate; the alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; and the alkali metal carbonate preferably includes sodium carbonate and / or potassium carbonate. The present application does not have special limitations on the amount of the dilute alkaline reagent aqueous solution, and the animal hide after the fourth depilation treatment can be immersed.

[0098] In the present application, the time of the fifth depilation treatment is preferably 4-6 hours, and in specific embodiments can be 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours.

[0099] In the present application, the depilation treatment preferably further includes: water washing the animal hide after the depilation treatment to obtain a depilated animal hide. In the present application, the number of times of water washing is preferably 3-5 times, and more preferably 4 times.

[0100] After obtaining the depilated animal hide, the present application performs a de-swelling treatment on the depilated animal hide to obtain a de-swollen animal hide.

[0101] In the present application, the de-swelling treatment preferably includes: adding a first portion of water and an inorganic salt to the depilated animal hide to perform a first de-swelling treatment, adding a second portion of water, adjusting the pH value to 4-4.5, adding an acid lipase to perform a second de-swelling treatment, and adding an acid protease to perform a third de-swelling treatment; the de-swelling treatment is preferably performed in a rotating drum; and the rotating speed of the rotating drum is preferably 5-12 r / min, and in specific embodiments 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 application, the mass-to-volume ratio of the pretreated animal hide to the first portion of water is preferably 1 kg: 0.8-1 L, and in specific embodiments can be 1 kg: 0.8 L, 1 kg: 0.85 L, 1 kg: 0.9 L, 1 kg: 0.95 L or 1 kg: 1 L.

[0103] In the present application, 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 hide, and in specific embodiments 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 application, the temperature of the first deswelling treatment is preferably 28-35°C, and in specific embodiments 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 50-60 min, and in specific embodiments can be 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min or 60 min; the end-point pH value of the first deswelling treatment is preferably 8-8.5, and in specific embodiments the end-point pH value can be 8, 8.1, 8.2, 8.3, 8.4 or 8.5.

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

[0106] In the present application, the mass of the acid lipase preferably accounts for 0.1-0.3% of the mass of the pretreated animal hide, and in specific embodiments 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 acid lipase is preferably 50-60 thousand units / g, and in specific embodiments can be 50 thousand units / g, 51 thousand units / g, 52 thousand units / g, 53 thousand units / g, 54 thousand units / g, 55 thousand units / g, 56 thousand units / g, 57 thousand units / g, 58 thousand units / g, 59 thousand units / g or 60 thousand units / g.

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

[0108] In the present application, the mass of the acid protease is 0.1-0.3% of the mass of the pretreated animal hide, and in specific embodiments 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 acid protease is preferably 8-10 million units / g, and in specific embodiments can be 8 million units / g, 8.1 million units / g, 8.2 million units / g, 8.3 million units / g, 8.4 million units / g, 8.5 million units / g, 8.6 million units / g, 8.7 million units / g, 8.8 million units / g, 8.9 million units / g, 9 million units / g, 9.1 million units / g, 9.2 million units / g, 9.3 million units / g, 9.4 million units / g, 9.5 million units / g, 9.6 million units / g, 9.7 million units / g, 9.8 million units / g, 9.9 million units / g or 10 million units / g.

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

[0110] In the present application, after the deswelling treatment, the deswelled animal hide is further preferably subjected to water washing to obtain a deswelled animal hide. In the present application, the number of times of water washing is preferably 3-5, and more preferably 4.

[0111] After obtaining the deswelled animal hide, the deswelled animal hide is subjected to acid swelling treatment in the present application to obtain an acid-swelled animal hide.

[0112] In the present application, the acid swelling treatment preferably comprises: adding water to the de-swollen animal hide, adjusting the pH value to 2.8-3.0, and standing after stirring the acid swelling treatment; 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 rotating speed of the rotating drum is preferably 5-12 r / min, and in specific embodiments can be 5 r / min, 6 r / min, 7 r / min, 8 r / min, 9 r / min, or 10 r / min, 11 r / min, or 12 r / min.

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

[0114] In the present application, the acid used for adjusting the pH value to 2.8-3.0 preferably comprises a formic acid solution and / or a sulfuric acid solution; the mass concentration of the formic acid solution is preferably 0.05-0.08%, and in specific embodiments 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 specific embodiments can be 0.08%, 0.09%, 0.010%, 0.011%, or 0.012%; when the acid is a mixed acid solution of a formic acid solution and a sulfuric acid solution, the volume ratio of the formic acid solution to the sulfuric acid solution in the mixed acid solution is preferably 1: 2-3, and more preferably 1: 2.5.

[0115] In the present application, the stirring time of the acid swelling treatment is preferably 30-60 min, and in specific embodiments can be 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0116] In the present application, the standing time of the acid swelling treatment is preferably 6-8 h, and in specific embodiments can be 6 h, 6.5 h, 7 h, 7.5 h, or 8 h.

[0117] In the present application, the acid swelling treatment further preferably comprises: washing the animal hide after the acid swelling treatment with water to obtain an acid-swollen animal hide. In the present application, the number of times of washing is preferably 3-5 times, and more preferably 4 times.

[0118] After obtaining the acid-swollen animal hide, the acid-swollen animal hide is crushed, the pH value is adjusted to 7-7.4, solid-liquid separation is performed, a solid component is obtained, the solid component is washed with water and then dried to obtain a medical-grade tissue-like natural collagen.

[0119] In the present application, the pulverization preferably comprises ball milling; the ball-to-material ratio of the ball milling is preferably 1:1-5, and in specific embodiments can be 1 kg:1-2 L, 1 kg:1-2 L.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 200-600 r / min, and in specific embodiments can be 200 r / min, 300 r / min, 400 r / min, 500 r / min or 600 r / min; the time of the ball milling is preferably 1-3 h, and in specific embodiments can be 1 h, 1.5 h, 2 h, 2.5 h or 3 h. In the present application, the grinding bodies used in the ball milling preferably comprise 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 can be 1 kg:1-2 L, 1 kg:1-2 L.5, 1:2, 1:2.5 or 1:3.

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

[0121] In the present application, the base used for adjusting the pH value to 7-7.4 preferably comprises one or more of hydroxides, carbonates and bicarbonates; the hydroxides preferably comprise one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide; the carbonates preferably comprise sodium carbonate and / or potassium carbonate; the alkali metal bicarbonates preferably comprise one or more of sodium bicarbonate, potassium bicarbonate and calcium bicarbonate; and in specific embodiments, the pH value can be 7, 7.1, 7.2, 7.3 or 7.4.

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

[0123] In the present application, the drying preferably comprises freeze-drying, and the present application is not particularly limited to the conditions of the freeze-drying.

[0124] In the present application, the drying is further preferably followed by sterilization of the dried natural collagen to obtain medical-grade tissue-like natural collagen. In the present application, the sterilization is preferably gamma-ray sterilization; the dose of the gamma-ray is preferably 6-25 KGy / h60Co, and in specific embodiments 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 / h60Co, 14 KGy / h60Co, 15 KGy / h60Co, 16 KGy / h60Co, 17 KGy / h60Co, 18 KGy / h60Co, 19 KGy / h60Co, 20 KGy / h60Co, 21 KGy / h60Co, 22 KGy / h60Co, 23 KGy / h60Co, 24 KGy / h60Co or 25 KGy / h60Co.

[0125] The preparation method provided by the present application has simple process, simple equipment, rich raw materials, low cost and easy scale-up industrial production. The medical-grade tissue-like natural collagen prepared by the present application has no degradation process of any natural collagen, and its basic composition is the existing form of collagen fibers in the tissue, which also has the spatial structure characteristics of the natural collagen in the tissue, so that it can more greatly imitate the natural tissue structure. The medical-grade tissue-like natural collagen prepared by the present application has more excellent mechanical properties, excellent enzyme / thermal stability and structural stability, low antigenicity and is easier to store, and has excellent hemostatic performance. Its high strength and toughness can meet the diversified requirements of the body on material performance, and it is an ideal collagen biomaterial. The medical-grade tissue-like natural collagen prepared by the present application is beneficial to the survival, proliferation and differentiation of cells, and can significantly promote the regeneration and repair of tissues and reduce the production of scars in wound and scar repair.

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

[0127] The application further provides application of the medical-grade tissue-like natural collagen in tissue engineering, regenerative medicine materials or implantable medical devices.

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

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

[0130] Example 1

[0131] A) Pretreatment of cowhide: the cowhide was repeatedly cleaned in a rotating drum to remove dirt on the cowhide, then soaked in water for 60 min after cleaning, and then subjected to mechanical meat cutting treatment to remove as much as possible subcutaneous fat, surface muscle, blood vessels and fascia tissue and other subcutaneous tissues on the cowhide, followed by cutting into small pieces of 5.0 mm x 5.0 mm, and then fully washed and soaked in normal saline at room temperature for 15 h to obtain pretreated cowhide;

[0132] B) Degreasing treatment of cowhide: 10 parts by weight of the pretreated cowhide was put into a rotating drum, 0.2 parts by weight of alkyl polyglycoside and 15 parts by volume of deionized water were added, and degreasing was carried out at 30℃ for 2 h, and then the degreased cowhide was repeatedly cleaned with warm water at 35℃ for 3 times to obtain degreased cowhide;

[0133] C) Hair removal treatment of cowhide: the degreased cowhide was placed in water with equal mass, 4 parts by weight of fatty alcohol polyoxyethylene ether and 3 parts by weight of ferrous sulfate were added to the water, and the rotating drum was rotated for 1.5 h, then 0.1 parts by weight of triethanolamine was added, and the rotating drum was continuously rotated for 10 min, then 0.35 parts by weight of sodium hydroxide was added, and the rotating drum was rotated for 15 min, then 0.6 parts by weight of hydrogen peroxide was added, and the rotating drum was rotated for 3 h, then the rotating drum was soaked in a 0.3% sodium hydroxide solution for 6 h, and then the degreased cowhide was repeatedly rinsed with ultrapure water for 3 times to obtain hair-removed cowhide;

[0134] D) Swelling treatment of the dehaired cowhide: the dehaired cowhide was put into a rotating drum, 8 parts by volume of ultrapure water was added, the temperature was controlled at 28°C, 0.05% by weight of sodium bisulfite was added, and the rotating was continued for 60 minutes. The final pH value was controlled at about 8. Then 10 parts by volume of deionized water and 3 parts by volume of formic acid were added into the rotating drum, the pH value was controlled at 4.5, and then 0.1% of acid lipase was added. After rotating for 20 minutes, 0.3% of acid protease was added, and the reaction was carried out at a constant temperature of 40°C for 2 hours. Finally, the swelling cowhide was obtained by repeatedly washing with ultrapure water for 3 times.

[0135] E) Acid swelling treatment of the dehaired cowhide: 10 parts by volume of ultrapure water was added into the swelling cowhide, and the pH value was adjusted to 2.8 by using a mixed acid solution of 0.05-0.08 wt% formic acid solution and 0.08-0.12 wt% sulfuric acid solution (volume ratio = 1:2). The mixture was stirred for 0.5 hours, and then was allowed to stand for 6 hours. Finally, the acid swelling cowhide was obtained by repeatedly washing with ultrapure water for 3 times.

[0136] F) Preparation of the tissue-like natural collagen: the acid swelling cowhide was put into a ball mill tank together with the grinding balls, so that the ball-to-material ratio was 1:1. The ball milling was carried out at a rotating speed of 200 r / min for 180 minutes. After the ball milling, the slurry was centrifuged at 15000 r / min for 15 minutes to remove the supernatant. The slurry was collected, and the pH value of the slurry was adjusted to 7.0 by using sodium hydroxide. The slurry was centrifuged at 15000 r / min for 15 minutes to collect the precipitate. The precipitate was washed with ultrapure water for 3 times. Finally, the medical grade tissue-like natural collagen was obtained by freeze-drying and sterilization by using γ-rays generated by a 60Co source at a dose of 6 KGy / h, and then was shaped and packaged.

[0137] Example 2

[0138] A) Pretreatment of the pigskin: the pigskin was repeatedly washed in a rotating drum to remove the dirt on the pigskin. After the washing, the pigskin was soaked in water for 90 minutes. Then, the pigskin was subjected to mechanical meat cutting treatment to remove the subcutaneous fat, surface muscle, blood vessels and fascia tissue and other subcutaneous tissues on the pigskin as much as possible. Then, the pigskin was cut into small pieces with a size of 8.0 mm x 8.0 mm. The small pieces were washed and soaked in physiological saline at room temperature for 13 hours to obtain the pretreated pigskin.

[0139] B) Degreasing treatment of the pigskin: 10 parts by weight of the pretreated pigskin was put into a rotating drum, 0.3 parts by weight of alkyl polyglycoside and 20 parts by volume of deionized water were added, and the degreasing treatment was carried out at a constant temperature of 35°C for 3 hours. After the degreasing treatment, the pigskin was repeatedly washed in warm water at 38°C for 4 times to obtain the degreased pigskin.

[0140] C) Pigskin depilation treatment: The above defatted pigskin was placed in an equal amount of water, 5 parts by weight of fatty alcohol polyoxyethylene ether and 4 parts by weight of ferrous sulfate were added to the water, 0.2 parts by weight of triethanolamine was added after 2 hours of rotation in a rotating drum, 0.4 parts by weight of sodium hydroxide was added after 15 minutes of continuous rotation, 0.7 parts by weight of hydrogen peroxide was added after 20 minutes of rotation, and the rotation was continued for 3.5 hours. After the end of the rotation, the pigskin was treated by immersion in a 0.5% by mass sodium hydroxide solution for 5 hours, and then rinsed repeatedly with ultrapure water for 4 times to obtain depilated pigskin;

[0141] D) Pigskin deswelling treatment: The above depilated pigskin was put into a rotating drum, 9 parts by volume of ultrapure water was added, the temperature was controlled at 30°C, and 0.12% of sodium bisulfite based on the weight of the pigskin was added, and the rotation was continued for 55 minutes, and the final pH value was controlled at about 8.3. After the end of the rotation, 10 parts by volume of deionized water and 3.5 parts by volume of formic acid were added to the same bath, the pH value was controlled at 4.2, and then 0.2% of acid lipase was added, and 0.2% of acid protease was added after 20 minutes of rotation, and the reaction was carried out at 40°C for 3 hours. Finally, the pigskin was rinsed repeatedly with ultrapure water for 4 times to obtain deswelled pigskin.

[0142] E) Pigskin acid swelling treatment: To the above deswelled pigskin, 15 parts by volume of ultrapure water was continuously added, and the pH value was adjusted to 2.9 by using a mixed acid solution of 0.05-0.08 wt% formic acid solution and 0.08-0.12 wt% sulfuric acid solution (volume ratio = 1:2), and the stirring was carried out for 0.8 hours. After standing for 7 hours, the pigskin was rinsed repeatedly with ultrapure water for 4 times to obtain acid-swelled pigskin.

[0143] F) Preparation of tissue-like natural collagen: The above acid-swelled pigskin was taken and put into a ball mill tank together with the grinding balls, so that the ball-to-material ratio was 1:3. The ball milling was carried out at a rotation speed of 400 r / min for 120 minutes. After the completion of the ball milling, the slurry was centrifuged at 16000 r / min for 13 minutes to remove the supernatant, and the slurry was collected. The pH value of the slurry was adjusted to 7.2 by using sodium carbonate, and the slurry was centrifuged at 16000 r / min for 13 minutes to collect the precipitate. The precipitate was washed with ultrapure water for 4 times. Finally, the precipitate was freeze-dried, sterilized by γ-rays generated by a 60Co with a dose of 18 KGy / h, and packaged to obtain medical-grade tissue-like natural collagen. In this embodiment, the grinding bodies used in the ball milling were large balls with a particle size of 12-13 mm and small balls with a particle size of 9-10 mm, and the number ratio of the large balls to the small balls was 1:2. In this embodiment, the rotation speed of the rotating drum was 10 r / min.

[0144] Example 3

[0145] A) Sheepskin pretreatment: The sheepskin was repeatedly cleaned in a rotating drum to remove dirt on the sheepskin, and then soaked in water for 120 min. The sheepskin was then mechanically deboned to remove as much subcutaneous fat, surface muscle, blood vessels and fascial tissue as possible. The sheepskin was then cut into small pieces of 10.0 mm x 10.0 mm, washed and soaked in physiological saline at room temperature for 10 h to obtain pretreated sheepskin;

[0146] B) Sheepskin degreasing treatment: 10 parts by weight of the pretreated sheepskin was placed in a rotating drum, 0.4 parts by weight of alkyl polyglycoside and 25 parts by volume of deionized water were added, and the degreasing was carried out at 37°C for 5 h. After degreasing, the sheepskin was repeatedly washed in warm water at 40°C for 5 times;

[0147] C) Sheepskin unhairing treatment: The degreased sheepskin was placed in an equal amount of water, 6 parts by weight of fatty alcohol polyoxyethylene ether and 5 parts by weight of ferrous sulfate were added to the water, and the sheepskin was rotated in the drum for 2.5 h. Then 0.3 parts by weight of triethanolamine was added, and the sheepskin was rotated for another 20 min. Then 0.45 parts by weight of sodium hydroxide was added, and the sheepskin was rotated for another 25 min. Then 0.8 parts by weight of hydrogen peroxide was added, and the sheepskin was rotated for another 4 h. After the end of the rotation, the sheepskin was soaked in a 0.8% sodium hydroxide solution for 4 h, and then rinsed with ultrapure water for 5 times;

[0148] D) Sheepskin swelling removal treatment: The unhairing sheepskin was placed in a rotating drum, 10 parts by volume of ultrapure water was added, the temperature was controlled at 35°C, and 0.2% of sodium bisulfite was added, and the sheepskin was rotated for 50 min. The final pH value was controlled at about 8.5. After the rotation was completed, 10 parts by volume of deionized water and 4 parts by volume of formic acid were added to the bath, and the pH value was controlled at 4. Then 0.3% of acid lipase was added, and the sheepskin was rotated for another 20 min. Then 0.1 of acid protease was added, and the reaction was carried out at 40°C for 5 h. Finally, the sheepskin was rinsed with ultrapure water for 5 times;

[0149] E) Sheepskin acid swelling treatment: To the above-mentioned swelling-removed sheepskin, 20 parts by volume of ultrapure water was added, and the pH value was adjusted to 3.0 with a mixed acid solution of 0.05-0.08 wt% formic acid solution and 0.08-0.12 wt% sulfuric acid solution (volume ratio = 1:2). The mixture was stirred for 1 h, and then allowed to stand for 8 h. Finally, the sheepskin was rinsed with ultrapure water for 5 times;

[0150] F) Preparation of tissue-like natural collagen: The above acid-swollen sheepskin was put into a ball mill tank with balls to make the ball-to-material ratio 1:5, and ball milling was carried out at a rotation speed of 600 r / min for 60 min. After the grinding, the slurry was centrifuged at 18000 r / min for 10 min, and the supernatant was removed. The slurry was collected, and the pH of the slurry was adjusted to 7.4 by sodium bicarbonate. The slurry was centrifuged at 18000 r / min for 10 min, and the precipitate was collected. The precipitate was washed with ultrapure water for 5 times. Finally, the precipitate was sterilized by freezing drying and gamma ray generated by a 60Co with a dose of 25 KGy / h, shaped and packaged to obtain medical grade tissue-like natural collagen. In this embodiment, the rotation speed of the rotating drum was 7 r / min.

[0151] Figure 1 The atomic force microscope (AFM) detection diagram of the medical grade tissue-like natural collagen prepared in Example 1 is shown in FIG. 1. As can be seen from FIG. 1, the prepared tissue-like natural collagen has a typical D-period light and dark cross structure of natural collagen, indicating that the original aggregate structure of the natural collagen is completely retained.

[0152] Comparative Example 1

[0153] The antigen-free collagen aggregate prepared in Example 2 of Chinese Patent CN104107456A.

[0154] Comparative Example 1 uses propyl and supercritical fluid degreasing. Acetone has certain toxicity, and the supercritical fluid device is more complex for industrialization. Under the same degreasing effect, the alkyl polyglycoside with better biocompatibility is used in the present application, and the rotating drum is also selected as the conventional equipment for leather, which is more suitable for industrial application. The preparation method provided by the present application adds a hair removal process compared with Comparative Example 1. This process can further remove non-collagen components such as impurities and fibrous stroma in the skin 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. In Comparative Example 1, a homogenizer is used for slurry process, and a large amount of small particles of skin pieces are inevitably retained due to the slurry process, which affects the subsequent application. The ball milling process used in the present application is more suitable for industrial production. The preparation period is shorter, the extraction rate of natural collagen is higher, and the sample prepared by the ball milling process is more uniform and the quality is more controllable.

[0155] Test Example 1

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

[0157] (1) The collagen extraction rate and impurity content of the collagen material are shown in Table 1.

[0158] Table 1 collagen extraction rate and impurity content results of collagen material

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

[0160] As shown in Table 1, compared with Comparative Example 1, the collagen extraction rate of the medical grade tissue-like natural collagen prepared in the application is significantly improved, and the impurity content is significantly reduced.

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

[0162] Table 2 Weight loss of collagen material in different temperature ranges

[0163]

[0164]

[0165] As shown in Table 2, compared with Comparative Example 1 and collagen, the medical grade tissue-like natural collagen prepared in the application has a lower weight loss rate in the temperature range of 50-800℃, 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 collagenase type I is dissolved in PBS buffer to prepare a collagenase degradation solution with a concentration of 2 mL / mg (1 U / mL), after freeze-drying treatment, the samples are immersed in the degradation solution for 30 days, and then the degradation experiment is detected in a 37℃ constant temperature biochemical incubator.

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

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

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

[0170] Table 3 Hemostatic, degradation and tensile strength results of collagen material

[0171] Sample Hemostatic time 30-day degradation rate Tensile strength Example 2 68s 68％ 3.2 MPa Comparative Example 1 116s 89％ 2.4 MPa Collagen 182s 100％ 0.9 MPa

[0172] As shown in Table 3, compared with Comparative Example 1 and collagen, the medical grade tissue-like natural collagen prepared in the application has a shorter hemostatic time and better hemostatic effect; a lower 30-day degradation rate and a slower degradation rate; and a higher tensile strength.

[0173] (4) MTT experiment

[0174] Experimental method: use the culture solution containing 10% fibroblast to prepare single cell suspension, inoculate 1000-10000 cells per hole to 96-hole plate, the volume of each hole is 200 μL. Under the standard culture condition, maintain the cell culture for 3-5 days. After the end of culture period, add MTT solution 10 μL per hole to continue incubation for 4 h, terminate the culture, carefully suck the culture supernatant in the hole, for the suspended cells, centrifugal after sucking the culture supernatant in the hole. Then, add 100 μL of dimethyl sulfoxide (DMSO) to each hole, shake for 10 min, so that the crystal is fully dissolved. Finally, select 490 nm wavelength, measure the light absorption value of each hole on the enzyme-linked immunosuppression instrument, and record the results. Draw the cell growth curve with time as the horizontal coordinate and the light absorption value as the vertical coordinate to evaluate the cell growth.

[0175] Figure 2 From the MTT experimental results of collagen, the antigen-free collagen aggregate prepared in Comparative Example 1 and the medical grade tissue-like natural collagen prepared in Example 2, it can be known that the medical grade tissue-like natural collagen prepared in the application provides a more suitable growth environment for cells and has a significant advantage 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 evenly draw a horizontal line on the back of a six-hole plate with the help of a ruler as a positioning reference for subsequent experiments; inoculate 3x10 5 cells per hole in the six-hole plate, and the total volume of each hole is 2 mL; after the cells are cultured for 24 h, use a 200 μL gun head to make a scratch on the cell layer along the pre-drawn line to form a "wound"; after the scratch is completed, the culture supernatant is sucked off, and the scratch is washed 2-3 times with PBS to remove the scratched cells; the old culture medium is discarded, and the culture medium containing the drug or serum-free culture medium is added according to the experimental grouping; the cells are placed in a 37°C, 5% CO2 incubator for culture. At the predetermined time points of 0 h, 24 h, etc., the cells are taken out, the change in the scratch width at the same position is observed under a microscope and recorded, and the change in cell migration is observed by taking photos.

[0178] Figure 3 The 24h scratch result graph of collagen, Figure 4 The 24h scratch result graph of the antigen-free collagen aggregate prepared in Comparative Example 1, Figure 5 The 24h scratch result graph of the medical grade tissue-like natural collagen prepared in Example 2, Figures 3 to 5 It can be known that, compared with collagen and the antigen-free collagen aggregate prepared in Comparative Example 1, the medical grade tissue-like natural collagen prepared in the application can promote cell growth and proliferation more, and is more conducive to tissue regeneration and scar reduction.

[0179] (6) HE staining of tissue

[0180] Figure 6 Animal skin tissue HE staining section pictures (x100) of medical grade tissue-like natural collagen prepared in Example 2 are shown in the following six pictures. As can be seen from the pictures, the collagen fiber bundles in the medical grade tissue-like natural collagen prepared in the application are clean and free of impurities, and the fibers remain intact, and the product is confirmed to be qualified.

[0181] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing medical-grade tissue-derived natural collagen, comprising the following steps: Remove the subcutaneous tissue from the animal skin, soak it in physiological saline, and obtain pretreated animal skin; The pretreated animal hide is then degreased to obtain degreased animal hide; The defatted animal hide is then subjected to a hair removal process to obtain a hairless animal hide; The hairless animal skin is subjected to a de-swelling treatment to obtain a de-swollen animal skin; The deswelling treatment includes: adding a first portion of water and inorganic salt to the dehaired animal skin for a first deswelling treatment; adding a second portion of water to adjust the pH to 4-4.5; adding acidic lipase for a second deswelling treatment; and adding acidic protease for a third deswelling treatment; the inorganic salt is sodium bisulfite; the temperature of the first deswelling treatment is 28-35℃, the time is 50-60 minutes, and the final pH value is 8-8.5; the time of the second deswelling treatment is 15-20 minutes; and the time of the third deswelling treatment is 2-5 hours. The de-swelled animal hide is subjected to acid swelling treatment to obtain acid-swelled animal hide; the acid swelling treatment includes: adding water to the de-swelled animal hide, adjusting the pH value to 2.8~3.0, stirring for acid swelling treatment, and then allowing it to stand for acid swelling treatment; the stirring acid swelling treatment time is 30~60 minutes; the standing acid swelling treatment time is 6~8 hours; The acid-swollen animal skin is crushed, the pH value is adjusted to 7-7.4, solid-liquid separation is performed to obtain a solid component, the solid component is washed with water and dried to obtain medical-grade tissue natural collagen.

2. The preparation method according to claim 1, characterized in that, The defatting process includes: adding a defatting agent and water to the pretreated animal skin for defatting; The mass ratio of the pretreated animal hide to the defatting agent is 1:0.01~0.04; The degreasing agent includes one or more of alkyl polyglycosides, phenacetin, laundry detergent, sodium lauryl sulfate, and fatty alcohol polyoxyethylene ether; The mass-to-volume ratio of the pretreated animal hide to water is 1 kg: 1.5~3 L; The degreasing treatment is carried out at a temperature of 30~37℃ for 2~5 hours. The degreasing process also includes washing with water.

3. The preparation method according to claim 1, characterized in that, The hair removal process includes: adding the defatted animal skin, water, polyoxyethylene ether and ferrous salt for a first hair removal process, adding an alcohol amine for a second hair removal process, adding an alkaline reagent for a third hair removal process, adding an oxidant for a fourth hair removal process, and adding a dilute alkaline reagent aqueous solution for a fifth hair removal process. The mass-to-volume ratio of the defatted animal skin to water is 1 kg: 1~2 L; The mass ratio of the pretreated animal skin to polyoxyethylene ether is 1:0.3~0.6; The mass ratio of the pretreated animal hide to ferrous salt is 1:0.3~0.6; The first hair removal treatment takes 90-150 minutes; The mass ratio of the pretreated animal hide to the alcohol amine is 1:0.01~0.03; The second hair removal treatment takes 10-20 minutes; The mass ratio of the pretreated animal skin to the alkaline reagent is 1:0.035~0.045; The third hair removal treatment takes 15-25 minutes; The mass ratio of the pretreated animal hide to the oxidant is 1:0.06~0.08; The fourth hair removal treatment takes 3-4 hours; The mass fraction of the dilute alkaline reagent aqueous solution is 0.3-0.8%; the time for the fifth hair removal treatment is 4-6 hours. The hair removal process also includes 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 ferrous ammonium sulfate; The alkanolamine includes one or more of triethanolamine, monoethanolamine and diethanolamine; The alkaline reagent includes alkali metal hydroxides and / or alkali metal carbonates; The oxidant includes one or more of hydrogen peroxide, sodium perborate, and sodium percarbonate; The alkaline reagent in the dilute alkaline aqueous solution includes alkali metal hydroxides and / or alkali metal carbonates.

5. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the pretreated animal hide to the first portion of water is 1 kg: 0.8~1 L; The inorganic salts comprise 0.05-0.2% of the mass of the pretreated animal hides; The mass-to-volume ratio of the pretreated animal hide to the second portion of water is 1 kg: 1~4 L; The acidic lipase accounts for 0.1-0.3% of the pretreated animal skin mass; the enzyme activity of the acidic lipase is 50,000-60,000 units / g. The temperature for the second deswelling treatment is 28~35℃; The acidic protease accounts for 0.1-0.3% of the pretreated animal skin mass; the enzyme activity of the acidic protease is 80,000-100,000 units / g. The temperature for the third deswelling treatment is 38~42℃; The swelling reduction treatment also includes washing with water.

6. The preparation method according to claim 1, characterized in that, During the acid swelling treatment, the mass-to-volume ratio of the pretreated animal skin to water is 1 kg: 1~2 L; The acid swelling treatment also includes washing with water.

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 200~600 r / min, and the time is 1~3 h; The drying includes 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 skins include the skins of one or more of the following animals: pigs, cattle, and sheep.

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

10. The use of the medical-grade tissue natural collagen as described in claim 9 in the preparation of regenerative medicine materials or implantable medical devices.

Images