A microfiber collagen hemostatic material and its preparation method and application

Abstract

The present invention discloses a method for preparing a microfiber collagen hemostatic material, which introduces anisotropic shear force and ultrasonic cavitation during the self-assembly process, utilizes mechanical disturbance to destroy the hydrogel scaffold between collagen molecules, and instantly strengthens the force between fiber units to precipitate microfiber collagen; the present invention also provides a method for preparing a microfiber collagen hemostatic material, which coordinates and regulates the collagen self-assembly preparation process to prepare a microfiber collagen hemostatic material with controllable fiber size. The present invention also provides a microfiber collagen hemostatic material and its application, which has a D period consistent with human tissue and a larger specific surface area, can efficiently and quickly adhere to platelets, trigger a coagulation mechanism, and achieve a rapid hemostatic effect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a microfiber collagen hemostatic material and a preparation method and application thereof. Background Art

[0002] It is difficult to stop bleeding from large areas caused by trauma, surgery and other factors by relying solely on the body's own coagulation mechanism. In order to cope with bleeding in various complex scenarios, ideal hemostatic materials should have the characteristics of rapid hemostasis, easy use, high safety, absorbability and promotion of wound healing. At present, the commonly used hemostatic materials mainly include molecular sieve hemostatic materials, chitosan, cellulose, collagen, etc. Among them, the hemostasis of chitosan lies in the fact that the chitosan ions with positive charges can adsorb red blood cells and platelets to form thrombi to achieve hemostatic effects. However, the hemostatic effect of chitosan itself is limited, and the hemostatic effect on severe bleeding trauma is general; cellulose is currently a widely used hemostatic material, but it will cause the local environment to become acidic during use. This highly acidic environment can cause neuronal degeneration. At the same time, cellulose belongs to the polysaccharide class, which may promote bacterial growth during degradation and lead to wound infection. In recent years, studies have found that collagen can promote platelet aggregation and has an inductive effect on fibroblasts, which can promote wound healing and can be used as a hemostatic material for rapid hemostasis of trauma.

[0003] At present, most collagen hemostatic materials are mainly freeze-dried sponges. Chinese Patent 201811641759.8 dissolves collagen in ionic liquid and obtains dry collagen sponge by wet spinning and solvent dehydration. The essence of collagen sponge is single-molecule collagen aggregates, and its microstructure is inconsistent with the collagen fiber morphology of human tissue. It has defects such as poor hemostatic effect, hard material, and poor wound adhesion performance. Chinese Patent 201410229105.4 discloses a method for self-assembly of aquatic collagen into fibers, and a hydrogel with D-periodic horizontal stripes is prepared. However, this type of gel material has a high water content and poor mechanical strength and cannot be used in the field of hemostasis. Chinese Patent 201710377072.1 discloses a method for preparing a microfiber collagen hemostatic material. This patent directly processes animal skin tissue by depilation, chopping, drying, and fluffing to obtain a skin fiber hemostatic material. Chinese patent 201811036592.7 disperses the skin pieces through ionic liquids, and uses the strong polarity of ionic liquids to freeze-dry to obtain fibrous collagen materials. The essence of the above two patent methods is to purify, dry, and crush animal skin tissue to prepare hemostatic materials. However, due to the tight weaving of skin tissue fibers, a large amount of fat, miscellaneous proteins, pigments and other substances in the skin cannot be completely removed. Ultimately, the obtained hemostatic material has low purity and poses a safety hazard. At the same time, there are a large number of terminal peptides between unextracted skin fibers, which may cause serious adverse reactions. In addition, the fiber size, fiber strength, and fiber stacking density of skin tissue are all affected by various factors such as the source species of animal skin, animal age, raw skin part difference, and animal growth environment, making the prepared collagen hemostatic material fiber size and phase uncontrollable, and there are huge differences in hemostatic performance. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a method for preparing a microfibril collagen hemostatic material, which can solve the problem of collagen self-assembly too quickly to form a strong gel and the problem of uncontrollable size of microfibril collagen. It can solve the problem that the current preparation process of collagen protein hemostatic materials has safety hazards and affects the collagen structure and biological activity.

[0005] The second object of the present invention is to provide a microfiber collagen hemostatic material, which can solve the problems of the current collagen hemostatic materials such as single fiber size and low purity, poor hemostatic performance, and poor compatibility with the human body.

[0006] The third object of the present invention is to provide an application of a microfiber collagen hemostatic material, which can solve the problems of slow triggering of the coagulation mechanism, low platelet adhesion efficiency and poor hemostatic effect during the hemostasis process.

[0007] The first object of the present invention is achieved by the following technical solutions:

[0008] A method for preparing a microfibril collagen hemostatic material comprises a raw material processing step, a collagen extraction step and a collagen self-assembly step; in the collagen self-assembly step, the collagen self-assembly system is simultaneously subjected to anisotropic stirring and ultrasonic treatment to self-assemble the collagen into a microfibril collagen precipitate.

[0009] Further, the following steps are included:

[0010] Raw material processing step: pre-treating the animal tissue, sterilizing and cleaning the pre-treated animal tissue, and preparing the processed animal tissue raw material;

[0011] Collagen extraction step: stirring and swelling the treated animal tissue raw material to obtain swollen animal tissue, adding pepsin to the swollen animal tissue for enzyme treatment, and filtering to obtain a collagen-containing supernatant;

[0012] Collagen self-assembly step: adding NaCl with a mass concentration of 1.5-2.5% to the collagen-containing supernatant to carry out collagen self-assembly, and simultaneously carrying out heterogeneous stirring treatment and ultrasonic treatment on the collagen self-assembly system to obtain microfibril collagen precipitate, thereby completing the preparation of microfibril collagen hemostatic material.

[0013] Furthermore, in the raw material processing step, the animal tissue is defatted, dehaired and deproteinized to obtain pre-treated animal tissue; the animal tissue is sourced from the skin or Achilles tendon tissue of cattle, pigs or sheep.

[0014] Furthermore, in the raw material processing step, the pretreated animal tissue is sterilized by immersing it in hydrogen peroxide with a mass concentration of 3-5% and a pH of 6.0-8.0; the material-liquid ratio of the pretreated animal tissue and hydrogen peroxide is 1:(20-60), and the immersion time is 4-6 hours.

[0015] Furthermore, in the collagen extraction step, the treated animal tissue material is stirred and swelled in an acetic acid solution with a concentration of 0.1-0.4M, the stirring and swelling time is 3-4 days, and the stirring and swelling temperature is 2-8°C; 1-3% of the wet weight of the tissue is added to the swollen animal tissue for enzyme treatment, the enzyme treatment time is 4-8h, and the enzyme treatment temperature is 8-15°C.

[0016] Furthermore, in the collagen self-assembly step, the pH of the collagen self-assembly system is 6.0-7.0, the collagen self-assembly temperature is 20-30° C., and the collagen self-assembly time is 1-4 hours.

[0017] Furthermore, in the collagen self-assembly step, the anisotropic stirring treatment is shear stirring during the collagen self-assembly process, the shear stirring speed is 50-100 rpm, and the direction of the shear stirring is changed every 25-35 minutes.

[0018] Furthermore, in the collagen self-assembly step, the ultrasonic treatment is performed for 15-25 minutes and then stopped for 15-25 minutes; the ultrasonic power is 5-15w.

[0019] The second object of the present invention is achieved by the following technical solutions:

[0020] A microfibril collagen hemostatic material is prepared by a method for preparing a microfibril collagen hemostatic material; the microfibril collagen of the microfibril collagen hemostatic material has a length of 2-10 mm; and the microfibril collagen has a D period.

[0021] The third object of the present invention is achieved by the following technical solutions:

[0022] The invention discloses an application of a microfiber collagen hemostatic material, wherein the microfiber collagen hemostatic material is applied to trauma hemostasis or surgical hemostasis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. A self-assembly method of microfiber collagen of the present invention increases spatial steric hindrance by acetic acid molecules in the raw materials, regulates other self-assembly conditions, slows down the speed of self-assembly, and does not form a strong gel; at the same time, anisotropic shear force and ultrasonic cavitation are used to respectively use mechanical disturbance to destroy collagen molecules to form a water-locking gel scaffold during the assembly process; ultrasonic cavitation forms cavitation bubbles between fiber units and quickly closes and ruptures, instantly strengthening the force between fiber units to form a microfiber collagen precipitate.

[0025] 2. The preparation method of a microfiber collagen hemostatic material of the present invention coordinates and regulates the collagen self-assembly preparation process to prepare a microfiber collagen hemostatic material with controllable fiber size. It is intended to achieve rapid adsorption and hemostasis of short microfiber collagen (2-5mm) to form blood clots; long microfiber collagen (5-10mm) to entangle blood clots to enhance the hemostatic effect; delay the addition of enzymes in the extraction stage to reduce the effect of enzymes on collagen structure and enhance the collagen self-assembly ability.

[0026] 3. The microfiber collagen hemostatic material of the present invention maintains the unique triple helix structure of collagen and is not modified by side chains, thus ensuring the biological activity of the material and promoting wound healing. The material is convenient and safe to use, controls endotoxin, is sterile, and has rapid hemostatic performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an AFM image of the self-assembled microfibers with D period in Example 1 of the present invention.

[0028] Figure 2 This is a micrograph of short fibers prepared in Example 2 of the present invention.

[0029] Figure 3 This is a micrograph of the long fibers prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] When the skin is damaged and bleeding occurs, the human body triggers its own physiological hemostasis mechanism. The exposure of damaged blood vessel wall tissue can activate the coagulation system in platelets and plasma. The activated platelets aggregate into groups and adhere to the wound to form soft thrombi to fill the wound. However, it is difficult to stop bleeding due to large-area bleeding caused by trauma, surgery and other factors by relying solely on the body's own coagulation mechanism. Conventional hemostasis techniques mainly include physical blocking, suturing, compression and other methods. However, due to the complexity of the wound surface, such as large-area, deep abdominal cavity, and organ bleeding, the above conventional hemostasis techniques cannot achieve rapid and effective hemostasis, which often causes bleeding shock and even endangers the patient's life. Therefore, high-performance hemostatic materials have become the key to rapid hemostasis and reducing mortality. In order to cope with bleeding in various complex scenarios, ideal hemostatic materials should have the characteristics of rapid hemostasis, easy use, high safety, absorbability and promotion of wound healing.

[0032] In recent years, studies have found that collagen can promote platelet aggregation and has an inductive effect on fibroblasts, which can promote wound healing and can be used as a hemostatic material for rapid hemostasis of trauma. Collagen is widely distributed in various animals and is the most important structural protein in animals, accounting for about 30% of the total protein in the body.

[0033] Collagen molecules have self-assembly function, which is a special biological activity. They can self-assemble in vivo and in vitro through hydrophobic bonds, hydrogen bonds and electrostatic interactions to form D-period collagen microfibers that are consistent with the tissue fiber structure in animals and have higher biological activity and biocompatibility.

[0034] A method for preparing a microfibril collagen hemostatic material comprises a raw material processing step, a collagen extraction step and a collagen self-assembly step; in the collagen self-assembly step, the collagen self-assembly system is simultaneously subjected to anisotropic stirring and ultrasonic treatment to self-assemble the collagen into a microfibril collagen precipitate.

[0035] Further, the following steps are included:

[0036] Raw material processing step: pre-treating the animal tissue, sterilizing and cleaning the pre-treated animal tissue, and preparing the processed animal tissue raw material;

[0037] Collagen extraction step: stirring and swelling the treated animal tissue raw material to obtain swollen animal tissue, adding pepsin to the swollen animal tissue for enzyme treatment, and filtering to obtain a collagen-containing supernatant;

[0038] Collagen self-assembly step: adding NaCl with a mass concentration of 1.5-2.5% to the collagen-containing supernatant to carry out collagen self-assembly, and simultaneously carrying out heterogeneous stirring treatment and ultrasonic treatment on the collagen self-assembly system to obtain microfibril collagen precipitate, thereby completing the preparation of microfibril collagen hemostatic material.

[0039] Furthermore, in the raw material processing step, the animal tissue is defatted, dehaired and deproteinized to obtain pre-treated animal tissue; the animal tissue is sourced from the skin or Achilles tendon tissue of cattle, pigs or sheep.

[0040] Furthermore, in the raw material processing step, the pretreated animal tissue is sterilized by immersing it in hydrogen peroxide with a mass concentration of 3-5% and a pH of 6.0-8.0; the material-liquid ratio of the pretreated animal tissue and hydrogen peroxide is 1:(20-60), and the immersion time is 4-6 hours.

[0041] Furthermore, in the collagen extraction step, the treated animal tissue material is stirred and swelled in an acetic acid solution with a concentration of 0.1-0.4M, the stirring and swelling time is 3-4 days, and the stirring and swelling temperature is 2-8°C; 1-3% of the wet weight of the tissue is added to the swollen animal tissue for enzyme treatment, the enzyme treatment time is 4-8h, and the enzyme treatment temperature is 8-15°C.

[0042] Furthermore, the collagen self-assembly step directly performs self-assembly by using the collagen supernatant obtained in the collagen extraction step, and the collagen supernatant contains acetic acid molecules. When acetic acid molecules exist in the system, steric hindrance is increased, other self-assembly conditions are simultaneously regulated, and the speed of self-assembly is slowed down.

[0043] Furthermore, in the collagen self-assembly step, the pH of the collagen self-assembly system is 6.0-7.0, the collagen self-assembly temperature is 20-30° C., and the collagen self-assembly time is 1-4 hours.

[0044] Furthermore, the assembly time is 1-2 hours or 2-4 hours.

[0045] Furthermore, when the assembly time is 1-2 hours, short fibers of 2-5 mm are obtained.

[0046] Furthermore, when the assembly time is 2-4 hours, long fibers of 5-10 mm are obtained.

[0047] Furthermore, in the collagen self-assembly step, the anisotropic stirring treatment is shear stirring during the collagen self-assembly process, the shear stirring speed is 50-100 rpm, and the direction of the shear stirring is changed every 25-35 minutes.

[0048] Further, the shear stirring speed may be between 50-60 rpm, between 60-70 rpm, between 70-80 rpm, between 80-90 rpm, or between 90-100 rpm. Preferably, the direction of the shear stirring is changed every 30 minutes.

[0049] Furthermore, in the collagen self-assembly step, the ultrasonic treatment is performed for 15-25 minutes and then stopped for 15-25 minutes; the ultrasonic power is 5-15W. The ultrasonic power can be between 5-7W, between 7-9W, between 9-11W, between 11-13W, and between 13-15W.

[0050] Furthermore, in a method for preparing a microfibrillar collagen hemostatic material, all reagents are of pharmaceutical grade or dry-baked in a 250° C. oven to remove endotoxins. All containers are soaked in 0.2-0.4M HCl or NaOH solution to remove endotoxins and sterilized by wet heat.

[0051] A microfibril collagen hemostatic material is prepared by a method for preparing a microfibril collagen hemostatic material; the microfibril collagen of the microfibril collagen hemostatic material has a length of 2-10 mm; and the microfibril collagen has a D period.

[0052] Furthermore, the D-period is that five triple-helical collagen molecules are arranged in a quarter staggered manner and highly oriented into microfibrous collagen with a D-period band-shaped space, and each D-period is 66±1.3 nm.

[0053] Furthermore, the microfiber collagen is divided into long fiber collagen or short fiber collagen; the long fiber collagen has a length of 5-10 mm; the short fiber collagen has a length of 2-5 mm.

[0054] The present invention is further described below in conjunction with specific implementation modes.

[0055] Example 1

[0056] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 3% hydrogen peroxide for 4 hours, with the material-liquid ratio of cowhide tissue to hydrogen peroxide being 1:20 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0057] The cleaned cowhide tissue was transferred to an acetic acid aqueous solution with a concentration of 0.1M for swelling, and the solid-liquid ratio of cowhide tissue to acetic acid aqueous solution was 1:40. The cowhide tissue swelling treatment time was 3 days, and the swelling temperature was controlled at 2-8°C. After swelling, pepsin at 1% of the wet weight of the tissue was added to the system, and the enzyme treatment time was 4h, and the enzyme treatment temperature was controlled at 8-15°C. Filter to obtain collagen supernatant.

[0058] The collagen supernatant was self-assembled, and 1.5% by mass of NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, and the shear stirring speed was 50 rpm. The stirring direction was changed every 25 minutes. Ultrasound was added at the same time, and the power of ultrasound was 5W. Ultrasound was stopped for 20 minutes after every 20 minutes of action. The total duration of self-assembly was 1 hour. Example 1 Preparation of AFM images of self-assembled microfibers of D period Figure 1 shown.

[0059] Example 2

[0060] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the ratio of cowhide tissue to hydrogen peroxide being 1:30 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0061] The cleaned cowhide tissue was transferred to an acetic acid aqueous solution with a concentration of 0.1M for swelling, and the solid-liquid ratio of the cowhide tissue to the acetic acid aqueous solution was 1:40. The cowhide tissue swelling treatment time was 3 days, and the swelling temperature was controlled at 2-8°C. After swelling, 2% of the wet weight of the tissue pepsin was added to the system, the enzyme treatment time was 4h, and the enzyme treatment temperature was controlled at 8-15°C. Filter to obtain the collagen supernatant.

[0062] The collagen supernatant was self-assembled, and 1.5% NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, and the shear stirring speed was 75 rpm. The stirring direction was changed every 25 minutes. Ultrasound was added at the same time, and the power of ultrasound was 10 W. Ultrasound was stopped for 20 minutes after every 20 minutes of action. The total duration of self-assembly was 2 hours. The short fibers prepared in Example 2 were as follows Figure 2 shown.

[0063] Example 3

[0064] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the ratio of cowhide tissue to hydrogen peroxide being 1:30 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0065] The cleaned cowhide tissue was transferred to an acetic acid aqueous solution with a concentration of 0.1M for swelling, and the solid-liquid ratio of the cowhide tissue to the acetic acid aqueous solution was 1:40. The cowhide tissue swelling treatment time was 3 days, and the swelling temperature was controlled at 2-8°C. After swelling, 3% of the wet weight of the tissue pepsin was added to the system, the enzyme treatment time was 4h, and the enzyme treatment temperature was controlled at 8-15°C. Filter to obtain the collagen supernatant.

[0066] The collagen supernatant was self-assembled, and 1.5% NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, the shear stirring speed was 100 rpm, and the stirring direction was changed every 25 minutes. Ultrasound was added at the same time, the power of ultrasound was 15W, and ultrasound was stopped for 20 minutes after every 20 minutes of action. The total duration of self-assembly was 2 hours.

[0067] Example 4

[0068] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the solid-liquid ratio of cowhide tissue to hydrogen peroxide being 1:40 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0069] The cleaned cowhide tissue was transferred to an acetic acid aqueous solution with a concentration of 0.25M for swelling, and the solid-liquid ratio of the cowhide tissue to the acetic acid aqueous solution was 1:40. The cowhide tissue swelling treatment time was 3 days, and the swelling temperature was controlled at 2-8°C. After swelling, 2% of the wet weight of the tissue pepsin was added to the system, the enzyme treatment time was 6 hours, and the enzyme treatment temperature was controlled at 8-15°C. Filter to obtain the collagen supernatant.

[0070] The collagen supernatant was self-assembled, and 2.0% NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, and the shear stirring speed was 75 rpm. The stirring direction was changed every 25 minutes. Ultrasound was added at the same time, and the power of ultrasound was 10 W. Ultrasound was stopped for 20 minutes after every 20 minutes of action. The total duration of self-assembly was 3 hours. The long fibers prepared in Example 4 are as follows Figure 3 shown.

[0071] Example 5

[0072] After degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 3% hydrogen peroxide for 4 hours, with the ratio of cowhide tissue to hydrogen peroxide being 1:40 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0073] The cleaned cowhide tissue was transferred to an acetic acid aqueous solution with a concentration of 0.25M for swelling, and the solid-liquid ratio of the cowhide tissue to the acetic acid aqueous solution was 1:40. The cowhide tissue swelling treatment time was 4 days, and the swelling temperature was controlled at 2-8°C. After swelling, 3% of the wet weight of the tissue pepsin was added to the system, the enzyme treatment time was 8h, and the enzyme treatment temperature was controlled at 8-15°C. Filter to obtain the collagen supernatant.

[0074] The collagen supernatant was self-assembled, and 2.0% NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, the shear stirring speed was 75rpm, and the stirring direction was changed every 25 minutes. Ultrasound was added at the same time, the power of ultrasound was 10W, and ultrasound was stopped for 20 minutes after every 20 minutes of action. The total duration of self-assembly was 4 hours.

[0075] Comparative Example 1

[0076] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the solid-liquid ratio of cowhide tissue to hydrogen peroxide being 1:40 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0077] First, the cowhide tissue was treated with pepsin, and the treatment conditions were the same as those in Example 4. Then, the tissue after enzyme treatment was subjected to swelling treatment, and the swelling treatment conditions were the same as those in Example 4. Filter to obtain collagen supernatant. The collagen supernatant was self-assembled, and 2.0% by mass of NaCl was added to the system. The pH of the self-assembly system was 6.0, and the self-assembly temperature was 25°C. During the self-assembly process, anisotropic shear stirring was performed, and the shear stirring speed was 75 rpm. The stirring direction was changed every 25 minutes, and the total self-assembly time was 3 hours.

[0078] Comparative Example 2

[0079] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the solid-liquid ratio of cowhide tissue to hydrogen peroxide being 1:40 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0080] First, the cowhide tissue was treated with pepsin, and the treatment conditions were the same as those in Example 4. Then, the tissue after enzyme treatment was subjected to swelling treatment, and the swelling treatment conditions were the same as those in Example 4. Filtering was performed to obtain collagen supernatant.

[0081] The collagen supernatant was self-assembled and added to the system with phosphate buffer solution at a ratio of 1:1. The pH of the self-assembly system was 7.0, the self-assembly temperature was 30°C, the total self-assembly time was 3h, and ultrasound was added at the same time. The ultrasound power was 10W, and the ultrasound was stopped for 20 minutes after every 20 minutes of action.

[0082] Comparative Example 3

[0083] After pretreatment of degreasing, dehairing and removing impurities and proteins, the cowhide tissue was immersed in 5% hydrogen peroxide for 4 hours, with the solid-liquid ratio of cowhide tissue to hydrogen peroxide being 1:40 and pH being 7.0 for sterilization. After immersion sterilization, the cowhide tissue was washed 3 times with injection water.

[0084] First, the cowhide tissue was treated with pepsin, and the treatment conditions were the same as those in Example 4. Then, the tissue after enzyme treatment was subjected to swelling treatment, and the swelling treatment conditions were the same as those in Example 4. Filtering was performed to obtain collagen supernatant.

[0085] The collagen supernatant was subjected to self-assembly, and 2.0% by mass of NaCl was added to the system. The pH of the self-assembly system was 6.0, the self-assembly temperature was 25° C., and the total self-assembly time was 3 h.

[0086] Performance Testing

[0087] 1. Physical properties of micro-collagen fiber hemostatic materials

[0088] Table 1 Product properties of Examples 1-5 and Comparative Examples 1-3

[0089] Microfiber physical properties Microfiber length (mm) Whether it has a three-dimensional structure Specific surface area (m2 / g) Example 1 2.53 yes 11.21 Example 2 3.15 yes 14.28 Example 3 4.34 yes 13.36 Example 4 5.18 yes 13.62 Example 5 7.36 yes 15.14 Comparative Example 1 6.37 no 8.63 Comparative Example 2 Gel formation yes / Comparative Example 3 Gel formation no /

[0090] As can be seen from Table 1, the microfiber collagen hemostatic materials of Examples 1-5 can maintain the three-dimensional structure of collagen, and the length of the formed microfibers is different according to the time difference of collagen self-assembly, and at the same time, they also have a larger specific surface area than Comparative Example 1. In Comparative Example 1, the collagen extraction was first added with enzymes and then swollen with acetic acid solution, so the formed microfibers could not maintain the unique spiral structure of collagen, but since anisotropic shear force was introduced for disturbance during the self-assembly process, no strong gel was formed; while in Comparative Example 2, the self-assembly process only included ultrasonic cavitation, and there was no disturbance of anisotropic shear force, so a gel with a certain strength was still formed, indicating that ultrasonic cavitation and anisotropic shear force have the effect of synergistically forming microfiber collagen. In Comparative Example 3, there was no anisotropic shear and ultrasonic cavitation in the collagen self-assembly, so the self-assembly formed a strong gel and no microfibers were generated.

[0091] 2. Hemostasis test

[0092] The microfiber collagen hemostatic materials of Examples 1-5 and Comparative Examples 1-2 were subjected to a hemostatic test, and the test method was as follows:

[0093] Take healthy adult New Zealand white rabbits, weighing about 2.0kg, regardless of gender. The experiment will test the hemostatic effect of the material on rabbit ears, 5 rabbits per group, shave the hair of each New Zealand rabbit's ears, and cut off the left ear vein, the right ear artery and two-thirds of the left hind limb femoral artery after disinfection with iodine. Take Examples 1-5 and Comparative Examples 1-2 as the experimental group, and the commercially available medical hemostatic dressing as the control group. When the wound blood emerges, first use ordinary gauze to absorb the blood, and then quickly apply the microfiber collagen hemostatic material of Examples 1-5 or Comparative Examples 1-2 to the wound, and start the stopwatch at the same time to observe the bleeding on the surface and around the hemostatic material, and use the ordinary gauze that has been accurately weighed to dip the bleeding every 20s, until the ordinary gauze can no longer dip into the bleeding, stop the stopwatch, record the time, and take the average value as the hemostasis time. The results are as follows:

[0094] Table 2 Microfiber collagen hemostatic performance test

[0095]

[0096] As can be seen from the above table, the hemostatic ability of the microfiber collagen hemostatic materials of Examples 1-5 of the present invention is stronger than that of Comparative Examples 1-2, and the hemostatic performance is improved by 47% compared with the medical dressings sold on the market. In Examples 1-5, the swelling and enzyme treatment of animal tissues in Examples 4 and 5 are more sufficient, more collagen is extracted, and the anisotropic shear frequency and ultrasonic cavitation intensity in the collagen self-assembly are improved, the time of collagen self-assembly is extended, and a gel with strength will not be formed, the tissue compatibility is better, and the long fibers formed can better achieve the hemostatic effect in a short time.

[0097] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a microfiber collagen hemostatic material, characterized in that: The method comprises a raw material processing step, a collagen extraction step and a collagen self-assembly step; in the collagen self-assembly step, the collagen self-assembly system is subjected to anisotropic stirring treatment and ultrasonic treatment at the same time, so that the collagen is self-assembled into microfibril collagen precipitates; The collagen extraction step includes stirring and swelling the treated animal tissue raw material to obtain swollen animal tissue, and adding pepsin to the swollen animal tissue for enzyme treatment.

2. The method for preparing a microfiber collagen hemostatic material according to claim 1, characterized in that: The following steps are involved: Raw material processing step: pre-treating the animal tissue, sterilizing and cleaning the pre-treated animal tissue, and preparing the processed animal tissue raw material; Collagen extraction step: stirring and swelling the treated animal tissue raw material to obtain swollen animal tissue, adding pepsin to the swollen animal tissue for enzyme treatment, and filtering to obtain a collagen-containing supernatant; Collagen self-assembly step: adding NaCl with a mass concentration of 1.5-2.5% to the collagen-containing supernatant to carry out collagen self-assembly, and simultaneously carrying out heterogeneous stirring treatment and ultrasonic treatment on the collagen self-assembly system to obtain microfibril collagen precipitate, thereby completing the preparation of microfibril collagen hemostatic material.

3. The method for preparing a microfiber collagen hemostatic material according to claim 2, characterized in that: In the raw material processing step, the animal tissue is defatted, dehaired and deproteinized to obtain pre-treated animal tissue; the animal tissue is sourced from the skin or Achilles tendon tissue of cattle, pigs or sheep.

4. The method for preparing a microfiber collagen hemostatic material according to claim 2, characterized in that: In the raw material processing step, the pretreated animal tissue is sterilized by soaking it in hydrogen peroxide with a mass concentration of 3-5% and a pH of 6.0-8.0; the material-liquid ratio of the pretreated animal tissue to hydrogen peroxide is 1:(20-60), and the soaking time is 4-6 hours.

5. The method for preparing a microfiber collagen hemostatic material according to claim 2, characterized in that: In the collagen extraction step, the treated animal tissue material is stirred and swelled in an acetic acid solution with a concentration of 0.1-0.4M, the stirring and swelling time is 3-4 days, and the stirring and swelling temperature is 2-8°C; 1-3% of the wet weight of the tissue pepsin is added to the swollen animal tissue for enzyme treatment, the enzyme treatment time is 4-8h, and the enzyme treatment temperature is 8-15°C.

6. The method for preparing a microfiber collagen hemostatic material according to claim 2, characterized in that: In the collagen self-assembly step, the pH of the collagen self-assembly system is 6.0-7.0, the collagen self-assembly temperature is 20-30° C., and the collagen self-assembly time is 1-4 hours.

7. The method for preparing a microfiber collagen hemostatic material according to claim 1, characterized in that: In the collagen self-assembly step, the anisotropic stirring treatment is to perform shear stirring during the collagen self-assembly process, the shear stirring speed is 50-100 rpm, and the direction of the shear stirring is changed every 25-35 minutes.

8. The method for preparing a microfiber collagen hemostatic material according to claim 1, characterized in that: In the collagen self-assembly step, the ultrasonic treatment is performed for 15-25 minutes and then stopped for 15-25 minutes; the ultrasonic power is 5-15w.

9. A microfiber collagen hemostatic material, characterized in that: The microfibril collagen hemostatic material is prepared by the preparation method of the microfibril collagen hemostatic material according to any one of claims 1 to 8; the microfibril collagen of the microfibril collagen hemostatic material has a length of 2-10 mm; and the microfibril collagen has a D period.

10. Use of the microfibrillar collagen hemostatic material according to claim 9 in the preparation of hemostatic materials.

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