A flocculent and fluffy collagen hemostatic material, and a preparation method and application thereof

Abstract

The present application provides a kind of flocculent fluffy collagen hemostatic material and its preparation method and application, it is related to the field of organic polymer compound processing, it is also related to medical material technical field.The preparation process of flocculent fluffy collagen hemostatic material is innovatively proposed in the present application, the preparation of flocculent collagen hemostatic material is realized by dry ice pore-forming-precursor induced self-assembly two-stage process;Snowy micro-porous mixture is formed in collagen solution under high-speed stirring state by using the super-fast cooling effect (-78.5 DEG C) of dry ice sublimation and CO2 bubble disturbance, and flocculent collagen precursor is obtained by freeze-drying and crushing;The crushed scaffold is used as "flocculent collagen precursor", in-situ epitaxial self-assembly is induced in PBS containing collagen, fluffy nanofibers are grown on the surface of precursor, and the final composite structure with fluffy core-fluffy boundary is formed by liquid nitrogen quick freezing and shaping, the material has the ability of rapid hemostasis;Dry ice pore-forming shortens the freezing cycle by 50%, and is safe and low in cost.

Description

Technical Field

[0001] This invention relates to the field of organic polymer compound processing, as well as the field of medical materials technology, and particularly to a flocculent, fluffy collagen hemostatic material, its preparation method, and its application. Background Technology

[0002] Collagen, as a major component of the natural extracellular matrix, is widely used in hemostatic materials due to its excellent biocompatibility, biodegradability, and procoagulant activity. Collagen hemostatic materials achieve rapid hemostasis through mechanisms such as activating platelet aggregation and promoting fibrin formation, while providing a three-dimensional scaffold for tissue repair. Compared with traditional hemostatic materials (such as gelatin sponges and fibrin glue), collagen raw materials have advantages such as low immunogenicity, controllable degradation rate, and good tissue adhesion, making them particularly suitable for hemostasis in irregular wounds and cavity bleeding scenarios.

[0003] Traditional collagen hemostatic products (such as sponges and membranes) achieve hemostasis through physical sealing and platelet activation, but they have limitations in filling deep wounds and rapid fluid absorption response. Currently, the main preparation methods for collagen hemostatic materials include freeze-drying, electrospinning, and cross-linking curing. However, these technologies have significant limitations: 1. Freeze-dried materials have low porosity, slow fluid absorption rate, and insufficient mechanical strength, and are prone to collapse in a wet state; 2. While electrospun materials have fine fibers, their dense structure and poor air permeability, along with complex processes and high costs; 3. Chemically cross-linked materials (such as those treated with glutaraldehyde) pose cytotoxic risks, while physically cross-linked materials (such as those irradiated with ultraviolet light) lack stability. Furthermore, existing materials are mostly in block or membrane form, making them unsuitable for hemostasis in deep wounds or minimally invasive surgeries. Some patents also directly freeze-dry animal tissue after mechanical dispersion, but due to the inability to completely remove a large number of non-collagenous impurities within the skin, the resulting hemostatic materials have low purity and pose safety risks.

[0004] The shortcomings of existing collagen hemostatic materials have been disclosed: 1. Freeze-dried collagen sponge materials obtained by direct freeze-drying tend to clump together, have a low specific surface area, and poor hemostatic effect; 2. Freeze-dried materials directly dispersed from animal tissues have safety hazards such as high impurity content; 3. Hemostatic materials obtained by electrospinning have a dense structure, poor water absorption, and low hemostatic efficiency; 4. Self-assembled collagen freeze-dried scaffolds have low structural strength and are prone to collapse when wet; 5. Cross-linked collagen hemostatic sponges have safety issues such as cross-linking agent residue. Summary of the Invention

[0005] To address the problems of caking, low specific surface area, and poor hemostatic effect in existing collagen hemostatic materials, this application provides a flocculent, fluffy collagen hemostatic material, its preparation method, and its application.

[0006] In a first aspect, this application provides a method for preparing a flocculent, loose collagen hemostatic material, comprising the following preparation steps:

[0007] Preparation of a slush-like collagen microporous mixture: Acidic collagen solution is rapidly stirred while adding fine dry ice particles of 2-7 mm in size until the collagen solution is completely frozen, thus obtaining a slush-like collagen microporous mixture; the weight ratio of collagen to dry ice in the acidic collagen solution is 1-1.5:2.5-3.2.

[0008] Preparation of flocculent collagen hemostatic material: After freeze-drying the snow-like collagen microporous mixture, flocculent collagen precursor was obtained; the flocculent collagen precursor was introduced into collagen-containing PBS buffer under stirring, and the system temperature was raised to perform self-assembly. After self-assembly, it was immediately and rapidly freeze-dried to obtain flocculent collagen hemostatic material with fluffy core-fur boundary.

[0009] The collagen concentration in the collagen-containing PBS buffer is 2-6 mg / mL.

[0010] Furthermore, the collagen concentration of the acidic collagen solution is 8-12 mg / mL.

[0011] Furthermore, in the preparation of the snow-like collagen microporous mixture, rapid stirring is performed at a speed of 100-500 rpm.

[0012] Furthermore, in the preparation of the snow-like collagen microporous mixture, rapid stirring is performed at a speed of 300-500 rpm.

[0013] Furthermore, in the preparation of the snow-like collagen microporous mixture, rapid stirring is performed using a ribbon-type stirring rod.

[0014] Furthermore, in the preparation of the flocculent collagen hemostatic material, the flocculent collagen precursor is mixed with PBS buffer at a ratio of 1:10 g / L.

[0015] Furthermore, the collagen-containing PBS buffer has a collagen concentration of 2-4 mg / mL, a PBS concentration of 0.01-0.05 M, and a pH of 6-8.

[0016] Furthermore, in the preparation of flocculent and fluffy collagen hemostatic materials, the self-assembly temperature is 30-40℃ and the self-assembly time is 8-28 min.

[0017] Furthermore, in the preparation of flocculent and fluffy collagen hemostatic material, the self-assembly temperature is 35-37℃ and the self-assembly time is 10-20 min.

[0018] Furthermore, in the preparation of the flocculent collagen hemostatic material, liquid nitrogen is used for rapid ice formation.

[0019] Furthermore, a method for preparing a flocculent, loose collagen hemostatic material includes the following steps:

[0020] Preparation of slush-like collagen microporous mixture: 3-5 L of acidic collagen solution with a concentration of 8-12 mg / mL was rapidly stirred using a ribbon stirrer at a speed of 300-500 rpm; while stirring, 80-100 g of fine dry ice particles with a size of 2-5 mm were added to the solution, and the addition was repeated every 20-40 s until the collagen solution was completely frozen, thus obtaining a slush-like collagen microporous mixture;

[0021] Preparation of flocculent, fluffy collagen hemostatic material: A slush-like collagen microporous mixture was freeze-dried and dispersed to obtain a flocculent collagen precursor. The flocculent collagen precursor was then introduced into a collagen-containing PBS buffer solution under stirring. 8-12 g of the flocculent collagen precursor was added to each 1 L of buffer solution. The collagen concentration in the PBS buffer solution was 2-4 mg / mL, the PBS concentration was 0.01-0.05 M, and the pH was 6-8. The system was heated to 35-37 ℃ to induce self-assembly. After self-assembly for 10-20 min, the mixture was immediately rapidly frozen with liquid nitrogen. After freeze-drying, a fluffy, fluffy collagen hemostatic material with a fluffy core and fibrous boundary was obtained.

[0022] Secondly, this application provides a flocculent, loose collagen hemostatic material, obtained using the preparation method described in this application.

[0023] Thirdly, this application provides an application of the aforementioned flocculent collagen hemostatic material in the preparation of hemostatic products, wherein the hemostatic product forms include flocculent powder hemostatic material, compressed columnar hemostatic material, and composite hydrogel hemostatic material.

[0024] Beneficial effects: 1. This invention innovatively proposes a preparation process for flocculent, fluffy collagen hemostatic materials. The process utilizes a two-stage process (first-stage pore creation + second-stage growth) involving dry ice pore formation and precursor-induced self-assembly. First-stage pore formation: Utilizing the ultra-rapid cooling effect of dry ice sublimation (-78.5 ℃) and CO2 bubble disturbance, a snow-like microporous mixture is formed in the collagen solution under high-speed stirring. After freeze-drying, the mixture is pulverized to obtain the flocculent collagen precursor. Second-stage growth: The pulverized scaffold serves as the "flocculent collagen precursor." Heating in collagen-containing PBS induces in-situ epitaxial self-assembly, growing fluffy nanofibers on the precursor surface. After rapid freezing with liquid nitrogen, a composite structure of fluffy core and fluffy boundary is finally formed. This material has a high specific surface area, increasing the contact area with platelets, and exhibits extremely high water absorption and swelling capacity, resulting in rapid hemostasis. Furthermore, it contains no cross-linking agents, and dry ice pore formation shortens the freezing cycle by 50%, making it safe and cost-effective.

[0025] 2. This application employs a dry ice sublimation foaming-freezing synergistic pore-forming method. Dry ice of a specific particle size is added to a collagen solution, and a high-speed ribbon-type stirring mechanism breaks down the bubbles generated by dry ice sublimation, achieving a microporous morphology after freeze-drying. The ribbon stirring ensures uniform circulation and stirring of the snow-like frozen collagen. Dry ice sublimation pore-forming and material pre-freezing into sand are achieved simultaneously. The resulting snow-like collagen microporous mixture has a uniform, dense, porous, and fluffy microstructure, resulting in a self-assembled hemostatic material with a higher specific surface area and a lower contact angle, thus achieving a superior hemostatic effect.

[0026] 3. The flocculent collagen hemostatic material obtained in this application has good resistance to enzymatic hydrolysis, and the complete degradation time in vivo can be controlled between 6 and 12 months; it has a high specific surface area, which increases the contact area with platelets, and has extremely high water absorption and swelling capacity, which enables rapid hemostasis; and it contains no cross-linking agent, and dry ice pore-forming shortens the freezing cycle by 50%, making it safe and low in cost.

[0027] 4. The flocculent collagen hemostatic material obtained in this application has multi-scenario adaptability and can be processed into various dosage forms such as flocculent powder, compressed columnar plugs, and composite hydrogels. It can be injected through tubing, compressed into shape, and compounded with drugs, and can be applied to rapid hemostasis in clinical practice in various departments. Attached Figure Description

[0028] Figure 1 These are comparative images of the appearance of the materials prepared in Example 1, Comparative Example 5, and Comparative Example 6; wherein, Figure 1 (a) in the text refers to the flocculent, fluffy collagen hemostatic material prepared in Example 1. Figure 1 (b) in the figure is the material appearance diagram of Comparative Example 5. Figure 1 (c) in the figure shows the appearance of the collagen hemostatic material prepared in Comparative Example 6;

[0029] Figure 2 These are photographs of the appearance of the snow-like collagen microporous mixture of Example 1, and microscopic morphology images of the snow-like collagen microporous mixtures obtained by the preparation methods of Examples 1-3 and Comparative Example 5.

[0030] Figure 3 These are microscopic morphology images of the slush-like collagen microporous mixtures obtained by the preparation methods of Examples 4, 5, Comparative Example 1 and Comparative Example 2 after freeze-drying.

[0031] Figure 4 These are magnified microscopic images of the hemostatic materials prepared in Examples 1-3, 6-9, and Comparative Examples 3-4.

[0032] Figure 5These are comparative graphs showing the rapid hemostatic effects of the hemostatic materials prepared in Examples 1-3, 5-9, and Comparative Examples 3-6. Detailed Implementation

[0033] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1: A flocculent, loose collagen hemostatic material and its preparation method, comprising the following preparation steps:

[0035] Preparation of the slush-like collagen microporous mixture: 4 L of an acidic collagen solution with a concentration of 8 mg / mL was placed in a beaker. The collagen solution was rapidly stirred using a ribbon stirrer at a speed of 300 rpm. While stirring, 100 g of fine dry ice particles with a size of 2 mm were added to the solution, and this was repeated every 30 s until the collagen solution was completely frozen, thus obtaining the slush-like collagen microporous mixture.

[0036] Preparation of flocculent, fluffy collagen hemostatic material: A slush-like collagen microporous mixture was freeze-dried and dispersed to obtain a flocculent collagen precursor. The flocculent collagen precursor was then poured into a collagen-containing PBS buffer (10 g of flocculent collagen precursor per 1 L of buffer) while stirring at 150 rpm (in other embodiments, the stirring speed can be set to any value between 100-200 rpm) (collagen concentration 4 mg / mL, 0.01 M PBS concentration, pH 6). The system was heated to 37 °C to allow self-assembly, and immediately after 10 min of self-assembly, it was rapidly frozen with liquid nitrogen. After freeze-drying, a fluffy, fluffy collagen hemostatic material with a fluffy core-flocculent boundary was obtained.

[0037] Example 2: A flocculent, loose collagen hemostatic material and its preparation method, comprising the following preparation steps:

[0038] Preparation of the slush-like collagen microporous mixture: 4 L of an acidic collagen solution with a concentration of 12 mg / mL was placed in a beaker. The collagen solution was rapidly stirred using a ribbon stirrer at a speed of 500 rpm. While stirring, 80 g of fine dry ice particles with a size of 5 mm were added to the solution, and this process was repeated every 30 s until the collagen solution was completely frozen, thus obtaining the slush-like collagen microporous mixture.

[0039] Preparation of flocculent, fluffy collagen hemostatic material: A slush-like collagen microporous mixture was freeze-dried and dispersed to obtain a flocculent collagen precursor. The flocculent collagen precursor was then poured into a collagen-containing PBS buffer (10 g of flocculent collagen precursor per 1 L of buffer) under stirring (collagen concentration 2 mg / mL, 0.01 M PBS concentration, pH 7). The system was heated to 37℃ for self-assembly, and immediately after 20 min of self-assembly, it was rapidly frozen with liquid nitrogen. After freeze-drying, a fluffy, fluffy collagen hemostatic material with a fluffy core-flocculent boundary was obtained.

[0040] Example 3: A flocculent, loose collagen hemostatic material and its preparation method, comprising the following preparation steps:

[0041] Preparation of the slush-like collagen microporous mixture: 4 L of an acidic collagen solution with a concentration of 10 mg / mL was placed in a beaker. The collagen solution was rapidly stirred using a ribbon stirrer at a speed of 400 rpm. While stirring, 90 g of fine dry ice particles with a size of 4 mm were added to the solution, and this process was repeated every 30 s until the collagen solution was completely frozen, thus obtaining the slush-like collagen microporous mixture.

[0042] Preparation of flocculent, fluffy collagen hemostatic material: A slush-like collagen microporous mixture was freeze-dried and dispersed to obtain a flocculent collagen precursor. The flocculent collagen precursor was then poured into a collagen-containing PBS buffer (10 g of flocculent collagen precursor per 1 L of buffer) under stirring (collagen concentration 3 mg / mL, 0.01 M PBS concentration, pH 8). The system was heated to 37 °C to allow self-assembly, and immediately after 15 min of self-assembly, it was rapidly frozen with liquid nitrogen. After freeze-drying, a fluffy, fluffy collagen hemostatic material with a fluffy core-flocculent boundary was obtained.

[0043] Example 4 discloses a flocculent, loose collagen hemostatic material and its preparation method, which differs from Example 1 in that an anchor-type stirring rod is used for stirring. This stirring method ensures thorough mixing of the material, but it cannot achieve uniform mixing of the resulting slush-like microporous mixture.

[0044] Example 5, a flocculent, loose collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the slush-like collagen microporous mixture, the stirring speed is lower, at 100 rpm. Uniform stirring cannot be achieved for the slush-like microporous mixture.

[0045] Example 6, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the flocculent collagen hemostatic material, the collagen concentration in the buffer solution is higher, at 6 mg / mL.

[0046] Example 7, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the flocculent collagen hemostatic material, the self-assembly time of the flocculent precursor is shorter, at 5 min.

[0047] Example 8, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the flocculent collagen hemostatic material, the self-assembly time of the flocculent precursor is longer, at 30 min.

[0048] Example 9, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the flocculent collagen hemostatic material, liquid nitrogen was not used for rapid freezing, but pre-frozen in a -20 ℃ refrigerator (requires 6 hours).

[0049] Comparative Example 1, a flocculent, fluffy collagen hemostatic material and its preparation method, differs from Example 1 in that the dry ice particles are larger, at 8 mm.

[0050] Comparative Example 2, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: in the preparation of the snow-like collagen microporous mixture, a larger amount of dry ice, 150 g, is used.

[0051] Comparative Example 3, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that the flocculent precursor is added to PBS buffer without stirring, and self-assembly occurs in a static state.

[0052] Comparative Example 4, a flocculent, fluffy collagen hemostatic material and its preparation method, differs from Example 1 in that: the flocculent precursor is self-assembled in physiological saline without the use of PBS buffer solution. The specific operation is as follows:

[0053] Take 4 L of an acidic collagen solution with a concentration of 8 mg / mL in a beaker and rapidly stir the collagen solution using a ribbon stirrer at a speed of 300 rpm. While stirring, add 100 g of fine dry ice particles with a size of 2 mm to the solution, repeating the addition every 30 s until the collagen solution is completely frozen, obtaining a slush-like collagen microporous mixture.

[0054] The slush-like collagen microporous mixture was freeze-dried and dispersed to obtain a flocculent collagen precursor. The flocculent collagen precursor was then poured into collagen-containing physiological saline solution (10 g of flocculent collagen precursor per 1 L of physiological saline solution) under stirring (collagen concentration 4 mg / mL, sodium chloride concentration 0.9%). The system was heated to 37 °C to allow self-assembly, and immediately after 10 min of self-assembly, it was rapidly frozen with liquid nitrogen. After freeze-drying, a fluffy, flocculent collagen hemostatic material with a fluffy core-fiber boundary was obtained.

[0055] Comparative Example 5, a flocculent collagen hemostatic material and its preparation method, differs from Example 1 in that: the slush-like collagen microporous mixture is directly and rapidly frozen with liquid nitrogen to prepare the final sample. It lacks the flocculent boundaries found in Example 5.

[0056] Comparative Example 6, a flocculent, loose collagen hemostatic material and its preparation method, differs from Example 1 in that: the preparation of a snow-like collagen microporous mixture is not performed, and the collagen solution is directly freeze-dried; the specific operation process is as follows:

[0057] Take 4 L of acidic collagen solution with a concentration of 8 mg / mL in a beaker, pour it into PBS buffer containing collagen while stirring (collagen concentration 4 mg / mL, 0.01M PBS concentration, pH 6), and heat the system to 37 °C to perform self-assembly. After self-assembly for 10 min, immediately freeze it with liquid nitrogen and freeze-dry to obtain collagen hemostatic material.

[0058] Experimental testing:

[0059] 1. Appearance and microstructure diagram:

[0060] By comparing the macroscopic morphological differences of the comparative examples in each embodiment, the influence of each key process on the final appearance of the hemostatic material was clarified. Using Example 1 as the basic process, the macroscopic appearance of two groups of samples were compared: Comparative Example 5 (the material has only a fluffy core, without flocculent fluffy boundaries) and Comparative Example 6 (the material is assembled solely through conventional solution self-assembly). The results are as follows:

[0061] Figure 1 The images show the appearance of the final hemostatic materials obtained by the preparation methods of Examples 1, 5, and 6. Figure 1 (a) in Example 1 shows the flocculent collagen hemostatic material prepared in Example 1, which has a fluffy, willow-like appearance, is loose overall, and does not clump. Figure 1 (b) is the material appearance diagram of Comparative Example 5. Since Comparative Example 5 directly freeze-dries the snow-like collagen microporous mixture without subsequent self-assembly process, the material has no flocculent and fluffy boundary and only shows a porous appearance. Therefore, the material is relatively hard and not fluffy. Figure 1 (c) shows the appearance of the collagen hemostatic material prepared in Comparative Example 6. The material exhibits a sheet-like structure rather than a flocculent and fluffy structure. This is because in the process of Comparative Example 6, collagen forms a collagen gel through conventional self-assembly. Therefore, the flocculent and fluffy appearance cannot be obtained after freeze-drying this material.

[0062] Figure 2 and Figure 3 The microstructure of the flocculent collagen precursor obtained by freeze-drying the snow-like collagen microporous mixture prepared in the examples and comparative examples is shown.

[0063] Figure 2 The middle image shows the appearance of the snow-like collagen microporous mixture of Example 1, and the microstructure of the snow-like collagen microporous mixtures prepared in Examples 1, 2, 3 and Comparative Example 5 after freeze-drying under the same conditions, observed by SEM at 300x magnification. Examples 1, 2, 3 and Comparative Example 5 used the same preparation process for the snow-like collagen microporous mixture, and the microstructure of their freeze-dried materials all exhibited a very dense porous and fluffy structure.

[0064] Figure 3 The images show the microstructure (magnification 300x) of the freeze-dried slush-like collagen microporous mixtures prepared in Examples 4-5 and Comparative Examples 1-2. In Example 4, a conventional anchor-type stirring paddle was used for stirring during the preparation of the slush-like collagen microporous mixture. Compared to a ribbon stirring rod, insufficient stirring uniformity during the freezing of the collagen solution into the slush-like collagen microporous mixture resulted in a precursor microstructure with numerous open pores. In Example 5, insufficient stirring speed led to the aggregation of carbon dioxide bubbles formed during the sublimation of dry ice, resulting in a larger pore size in the microstructure. In Comparative Example 1, the added dry ice particles were too large, resulting in a sheet-like layered structure of the precursor microstructure after freeze-drying, with no obvious pores. Comparative Example 2 used a large amount of dry ice, ultimately leading to a large number of cavities in the material. Because the above four comparative examples did not form a precursor material with a dense porous structure, they did not obtain a hemostatic material with a fluffy appearance after the subsequent self-assembly process.

[0065] Figure 4 The microscopic images are magnified microscopic images of the hemostatic materials prepared in the examples and comparative examples. The final material was placed on a glass slide, and physiological saline was added to disperse it before observation (100 mg of sample was taken for each example and comparative example, and 2 g of physiological saline was added to disperse it). The magnification was 50 times.

[0066] from Figure 4It can be seen that: the three groups of flocculent collagen hemostatic materials in Examples 1-3 were uniformly dispersed in physiological saline, and their outer surfaces showed obvious boundaries of numerous fine fiber filaments, indicating that the hemostatic materials prepared by this process have a fluffy core and a fibrous flocculent boundary structure. In Example 6, the collagen concentration in PBS was high during the self-assembly process, resulting in a high speed and degree of collagen fiber formation between the flocculent collagen precursors, and the final hemostatic material had a distinctly robust fiber appearance; the self-assembly time of the hemostatic material in Example 7 was relatively short, resulting in indistinct flocculent fiber boundaries and relatively aggregated material; in Example 8, due to the excessively long self-assembly time, the fiber filaments formed between the collagen flocculent precursors adhered severely, forming a large, diffuse microstructure of the hemostatic material; in Example 9, because rapid crystallization freezing with liquid nitrogen was not used, it slowly froze at -20℃, resulting in larger ice crystal particles, and the material after freeze-drying had relatively large pores, leading to severe direct aggregation of the hemostatic material. In Comparative Example 3, because no stirring was used during the self-assembly process, the static assembly process caused the fibers produced by the flocculent collagen precursor to link together, resulting in a compact flocculent material without loose pores and fiber boundary structure. In Comparative Example 4, because no PBS buffer system was used during the self-assembly process, the acidic flocculent collagen precursor buffered the pH of the system to acidity in physiological saline, inhibiting the self-assembly process. This resulted in a hemostatic material with a sponge structure, without porous structure and flocculent boundaries.

[0067] 2. Specific surface area determination:

[0068] The higher the specific surface area of ​​a hemostatic material, the stronger its blood absorption capacity, the greater the probability of contact with platelets, and the higher the probability of activating platelets to initiate the coagulation cascade reaction. The specific surface area of ​​the hemostatic material samples in each example was determined according to the "Specific Surface Area Determination Method" in General Chapter 0991 of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The experimental results are shown in the table below.

[0069] Table 1: Specific surface area of ​​hemostatic materials prepared by various processes

[0070]

[0071] Table 1 shows the specific surface area results of the collagen hemostatic materials prepared in each embodiment and comparative example. As can be seen from Table 1:

[0072] The specific surface area of ​​Examples 1-3 is all above 170 m² / g, indicating that the collagen hemostatic material prepared in these examples has a larger contact area with blood during hemostasis, higher efficiency in triggering platelet coagulation cascade reaction, and faster hemostasis speed.

[0073] The collagen hemostatic materials prepared in Examples 4, 5, Comparative Examples 1, and 2 had poor uniformity due to differences in stirring method, stirring speed, dry ice particles, and dry ice dosage during the preparation of the slush-like collagen microporous mixture. This resulted in uneven growth of flocculent collagen fibers at the boundaries during subsequent self-assembly, leading to partial collapse structures and thus a lower specific surface area compared to the examples.

[0074] In Example 6, the collagen concentration in PBS was high during the self-assembly process, resulting in coarser collagen flocculent fibers formed at the boundary, which led to a decrease in the specific surface area of ​​the material.

[0075] The self-assembly time of the hemostatic materials prepared in Examples 7 and 8 was not optimal, resulting in the flocculent fibers formed at the pore boundaries being either too thin or too thick, leading to a decrease in specific surface area.

[0076] Example 9 did not use liquid nitrogen for rapid crystallization freezing; instead, it slowly iced at -20 °C, resulting in larger ice crystal particles. After freeze-drying, the material had relatively larger pores, and the specific surface area decreased accordingly.

[0077] In Comparative Example 3, because no stirring was carried out during the self-assembly process, the static assembly process caused the fibers produced by the flocculent collagen precursor to link together, resulting in a reduction in the boundary conducive to collagen fiber growth after freeze-drying, thus reducing the specific surface area.

[0078] Comparative Example 4 did not use a PBS buffer system during the self-assembly process, which caused the acidic flocculent collagen precursor to buffer the pH of the system to acidity in physiological saline, inhibiting the self-assembly process and ultimately resulting in a sponge structure with a very low specific surface area for the hemostatic material.

[0079] Comparative Example 5 only freeze-dried the snow-like collagen microporous mixture without subsequent self-assembly. Therefore, there were no fine fiber flocs between the micropores, resulting in the lowest specific surface area for this sample.

[0080] Comparative Example 6 is a hemostatic material prepared by directly self-assembling collagen. Since the microstructure of this material has no flocculent extensions and no sheet-like structure, the specific surface area of ​​the material is relatively low.

[0081] 3. Contact angle measurement:

[0082] The hydrophilicity of the prepared collagen hemostatic material was determined by contact angle. When a droplet reaches equilibrium on a solid surface, the angle (θ) between the tangent of the liquid-gas interface and the solid-liquid interface at the gas-liquid-solid three-phase interface is considered. A smaller θ indicates more complete liquid spread and stronger hydrophilicity; a larger θ indicates greater liquid contraction and stronger hydrophobicity. In this experiment, the collagen hemostatic material prepared in the examples and comparative examples was pressed into a sheet of uniform thickness at 5 kPa, with the thickness controlled at 1.0 ± 0.2 mm. The contact angle of simulated body fluid SBF (pH 7.4) and whole blood plasma (heparin anticoagulated, centrifuged and supernatant plasma) was measured using a contact angle meter. The liquid volume was set to 2 μL. The contact angle was measured within 5 seconds of the droplet contacting the surface, and the measurement was repeated n=5 times (at different locations). The contact angle results for each example and comparative example are shown in Table 2 below.

[0083] Table 2: Contact angles of hemostatic materials prepared by various processes

[0084]

[0085] The hydrophilicity of a material is closely related to its contact angle; a lower contact angle indicates stronger hydrophilicity and a higher hemostatic effect. Table 2 shows that:

[0086] The contact angles of the simulated body fluids and plasma in Examples 1-3 were all lower than those in all comparative examples, indicating that the flocculent and fluffy collagen hemostatic material prepared by the process in these examples has good hemostatic properties.

[0087] The hemostatic materials prepared in Examples 4, 5, Comparative Examples 1, and 2 had lower porosity in the flocculent collagen precursors due to differences in stirring method, stirring speed, dry ice particles, and dry ice dosage during the preparation of the snow-like collagen microporous mixture. Furthermore, the growth and shrinkage of collagen fiber boundaries during self-assembly resulted in a significant increase in the contact angle of the simulated body fluids and plasma in these four comparative examples compared to the examples, along with decreased uniformity and weakened hydrophilicity.

[0088] The hemostatic material obtained in Example 6 was relatively hard and had poor hydrophilicity, possibly because it underwent self-assembly with high concentrations of collagen, resulting in coarse fibers in the final material.

[0089] The increased contact angle and decreased hydrophilicity of the hemostatic materials obtained in Examples 7 and 8 may be due to the fact that the self-assembly time of the hemostatic materials was not optimal, resulting in the flocculent fibers formed at the pore boundaries being too thin or too thick.

[0090] In Example 9, the pre-freezing process was too hot, resulting in larger ice crystal particles, which led to larger pores in the material and reduced hydrophilicity.

[0091] The hemostatic material obtained in Comparative Example 3 had poor hydrophilicity and a significantly increased contact angle. This may be because it was self-assembled in a static state, and the raw materials were relatively compacted before freeze-drying, lacking flocculent and loose boundaries.

[0092] The contact angle of the hemostatic material obtained in Comparative Example 4 was significantly increased and showed obvious unevenness, possibly due to the need to achieve the optimal pH for self-assembly.

[0093] The hemostatic material obtained in Comparative Example 5 had the largest contact angle and the lowest hydrophilicity, possibly because only the snow-like collagen microporous mixture was freeze-dried without subsequent self-assembly, and no fine fiber flocs were present between the micropores.

[0094] The material in Comparative Example 6 has poor hydrophilicity, possibly because the hemostatic material was prepared by directly self-assembling collagen. Since the microstructure of this material does not have flocculent extensions, it has a sheet-like structure.

[0095] 4. Swelling rate determination:

[0096] The absorption efficiency of collagen hemostatic materials during rapid hemostasis was evaluated by assessing the swelling rate of the collagen hemostatic material in physiological saline. Approximately 1g of the collagen hemostatic material prepared in each example and comparative example was weighed and placed in a 500-mesh stainless steel sieve. The stainless steel sieve was immersed in physiological saline and allowed to stand for 60 seconds to ensure the hemostatic material fully absorbed the saline. The sieve was then removed, drained, and weighed to obtain the mass of the collagen hemostatic material after water absorption. The swelling rate was calculated using the formula: water absorption swelling rate = (W...) / (W...) 湿 - W 干 ) / W 干 ×100%. Five parallel measurements were taken for each sample group, and the RSD was calculated. The experimental results are shown in Table 3.

[0097] Table 3 Swelling rate of hemostatic materials prepared by various processes

[0098]

[0099] The average swelling rate of the flocculent collagen hemostatic materials prepared in Examples 1-3 was higher than that of all comparative examples, with a swelling rate of more than 2400%, indicating that the material can quickly absorb blood after contact with blood and trigger the platelet coagulation reaction.

[0100] The collagen hemostatic materials prepared in Examples 4, 5, Comparative Examples 1 and 2 had large and uneven pores in their cores before self-assembly due to differences in stirring method, stirring speed, dry ice particles, and dry ice dosage during the preparation of the snow-like collagen microporous mixture. This resulted in a significant increase in swelling rate and large deviations in experimental results.

[0101] Example 6: Due to the high concentration of collagen in the solution, the resulting fiber boundaries are relatively coarse, hydrophilicity is reduced, and swelling rate is low.

[0102] The self-assembly time of the hemostatic materials in Examples 7 and 8 was not optimal, resulting in the flocculent fibers formed at the pore boundaries being either too thin or too thick, leading to a significant reduction in swelling degree.

[0103] In Example 9, because the pre-freezing temperature was too high, the ice crystal particles formed were larger, resulting in larger material pores and thus a reduced degree of swelling.

[0104] In Comparative Example 3, due to self-assembly under static conditions, the raw materials of the adhesive were relatively compacted and had smaller pores before freeze-drying, resulting in a decrease in swelling rate.

[0105] Comparative Example 4 had poor assembly results due to self-assembly conditions deviating from the optimal state, resulting in a very low swelling rate of the obtained material.

[0106] Comparative Example 5 only freeze-dried the snow-like collagen microporous mixture without subsequent self-assembly. Therefore, there were no fine fiber flocs between the micropores, so the swelling degree of this sample was the lowest among all samples.

[0107] Comparative Example 6 was prepared by directly self-assembling collagen to create a hemostatic material. Since the material's microstructure lacked flocculent extensions and sheet-like structures, its swelling degree was poor.

[0108] 5. Rabbit femoral artery hemostasis experiment:

[0109] Based on the macro / microstructure, specific surface area, contact angle, and swelling rate of the hemostatic materials mentioned above, Examples 1-3, Examples 5-9, and Comparative Examples 3-6 were selected as samples for the rapid hemostasis experiment of rabbit femoral arteries. 1g of sample was used to cover and apply pressure-free hemostasis to severed rabbit femoral arteries. The rapid hemostatic ability of the material was determined by observing the hemostasis time on the femoral artery. Specific hemostasis procedures were as follows: Figure 5 .

[0110] The results of the rabbit femoral artery hemostasis experiment show that the flocculent collagen hemostatic material prepared in Examples 1-3 did not show any bleeding during the non-pressure hemostasis process of the severed rabbit femoral artery, and 1g of hemostatic material was confirmed to have completed hemostasis in 2 minutes and 9 seconds and 2 minutes and 10 seconds, indicating high rapid hemostasis efficiency.

[0111] In Example 2, no bleeding was observed after 2 minutes and 25 seconds when 1g of material was not completely poured onto the blood vessel during the hemostasis process, and no bleeding was observed after 3 minutes and 11 seconds, indicating that the hemostatic performance of the material prepared in Example 2 was comparable to that in Examples 1 and 3.

[0112] In Example 5, bleeding was still occurring at 9 minutes and 42 seconds, indicating that the material prepared by continuing to work downwards in a non-uniform state of the snow-like collagen microporous mixture lost its ability to stop bleeding quickly due to insufficient core fluffiness.

[0113] In Example 6, hemostasis was achieved after 3 minutes and 15 seconds of observation, with a slightly worse hemostatic effect than in Example 6, indicating that the collagen concentration in the solution during the self-assembly process should not be too high.

[0114] In Examples 7, 8, and 9, hemostasis was achieved at 5 minutes 31 seconds, 4 minutes 53 seconds, and 5 minutes 07 seconds, respectively. This indicates that a shorter self-assembly time, a longer self-assembly time, and a higher pre-freezing temperature all affect the flocculent fluffiness of the final material, thus reducing its hemostasis efficiency.

[0115] Comparative Examples 3 and 4 still showed significant bleeding at 8 minutes and 22 seconds and 6 minutes and 59 seconds, respectively, indicating that the hemostatic materials obtained by static self-assembly and self-assembly under suboptimal conditions during the preparation process do not have the ability to stop bleeding quickly, possibly due to insufficient boundaries of their fluffy, fibrous fibers.

[0116] Comparative Example 5 still exhibited significant bleeding at 6 minutes and 30 seconds, indicating that the material's ability to rapidly stop bleeding was compromised by having only a fluffy core and lacking the flocculent fibrous boundary formed by subsequent self-assembly.

[0117] Comparative Example 6 showed bleeding from the blood vessels at 9 minutes and 20 seconds of observation, indicating that the hemostatic material obtained by simply performing collagen self-assembly has poor hemostatic performance.

[0118] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a flocculent and bulky collagen hemostatic material, characterized by, The preparation method comprises the following steps: Preparation of the snow-like collagen microporous mixture: under the condition of rapid stirring, fine granular dry ice with a size of 2-7 mm is added to the acid collagen solution until the collagen solution is completely iced, and a snow-like collagen microporous mixture is obtained; the weight ratio of collagen in the acid collagen solution to dry ice is 1-1.5:2.5-3.2; Preparation of the flocculent fluffy collagen hemostatic material: after the snow-like collagen microporous mixture is freeze-dried, a flocculent collagen precursor is obtained; and the flocculent collagen precursor is introduced into a collagen-containing PBS buffer under stirring, and the system temperature is warmed for self-assembly; immediately after self-assembly, rapid ice formation is performed for freeze-drying, and a flocculent fluffy collagen hemostatic material with a fluffy core-fleece boundary is obtained; The collagen concentration of the collagen-containing PBS buffer is 2-6 mg / mL. In the preparation of the snow-like collagen microporous mixture, the rapid stirring is at a speed of 300-500 rpm.

2. The method of claim 1, wherein the method is characterized by, The collagen concentration of the acid collagen solution is 8-12 mg / mL.

3. A process for the preparation of a flocculent and bulky collagen hemostatic material according to any one of claims 1-2, characterized in that, In the preparation of the snow-like collagen microporous mixture, the rapid stirring adopts a screw ribbon stirring rod.

4. The method of claim 3, wherein the method is characterized by, In the preparation of the flocculent fluffy collagen hemostatic material, the flocculent collagen precursor is mixed with the PBS buffer at a ratio of 1:10 g / L.

5. A process for the preparation of a flocculent and bulky collagen hemostatic material according to any of claims 1-2, 4, characterized in that, The collagen concentration of the collagen-containing PBS buffer is 2-4 mg / mL, the PBS concentration is 0.01-0.05 M, and the pH is 6-8.

6. The method of claim 5, wherein the method further comprises the step of adding a gelling agent to the collagen solution. In the preparation of the flocculent fluffy collagen hemostatic material, the self-assembly temperature is 30-40℃, and the self-assembly time is 8-28 min.

7. The method of claim 6, wherein the method further comprises the step of adding a gelling agent to the collagen solution. In the preparation of the flocculent fluffy collagen hemostatic material, the self-assembly temperature is 35-37℃, and the self-assembly time is 10-20 min.

8. A process for the preparation of a flocculent and bulky collagen hemostatic material according to any one of claims 1-2, 4, 6-7, characterized by, In the preparation of the flocculent fluffy collagen hemostatic material, the rapid ice formation adopts liquid nitrogen rapid ice formation.

9. The method of claim 8, wherein the method further comprises the step of adding a gelling agent to the collagen solution. The preparation method comprises the following steps: Preparation of the snow-like collagen microporous mixture: 3-5 L of an acid collagen solution with a concentration of 8-12 mg / mL is rapidly stirred using a screw ribbon stirring rod at a speed of 300-500 rpm; under the condition of stirring, 80-100 g of fine granular dry ice with a size of 2-5 mm is added to the solution, and the addition is repeated every 20-40 s until the collagen solution is completely iced, and a snow-like collagen microporous mixture is obtained; Preparation of the flocculent fluffy collagen hemostatic material: after the snow-like collagen microporous mixture is freeze-dried and dispersed, a flocculent collagen precursor is obtained; and the flocculent collagen precursor is introduced into a collagen-containing PBS buffer under stirring, 8-12 g of the flocculent collagen precursor is added per 1 L of the buffer, the collagen concentration of the collagen-containing PBS buffer is 2-4 mg / mL, the PBS concentration is 0.01-0.05 M, the pH is 6-8, and the system temperature is warmed to 35-37℃ for self-assembly; after self-assembly for 10-20 min, liquid nitrogen is used for rapid ice formation, and after freeze-drying, a flocculent fluffy collagen hemostatic material with a fluffy core-fleece boundary is obtained.

10. A fibrous, fluffy collagen hemostatic material, characterized in that, The preparation method is obtained by any one of claims 1-9.

11. Use of the fibrous, fluffy collagen hemostatic material according to claim 10 for the preparation of a hemostatic product, characterized in that, The form of the hemostatic product includes a flocculent powder hemostatic material, a compressed columnar plug hemostatic material, and a composite hydrogel hemostatic material.

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