A composite pore structure collagen hemostatic sponge and its preparation method

Abstract

The present invention discloses a composite pore structure collagen hemostatic sponge and a preparation method thereof, which relates to the technical field of biomedical materials. The preparation method comprises the following steps: (1) dissolving collagen in ultrapure water to prepare solution B and solution S of different concentrations; (2) adding solution B and solution S into a mold according to different schemes for directionally freezing and freeze-drying to obtain a precursor sponge; (3) adding EDC and NHS into a MES ethanol buffer solution to obtain an EDC / NHS ethanol solution; (4) soaking the precursor sponge in the EDC / NHS ethanol solution for cross-linking, washing and freeze-drying to obtain a composite pore structure collagen hemostatic sponge. The hemostatic sponge has excellent unilateral or bilateral bleeding blocking ability and shape memory function, solves the problems of existing hemostatic materials in the non-compression hemostasis process, such as slow hemostasis speed and mismatch between degradation and wound healing, and has great application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a collagen hemostatic sponge with a composite pore structure and a preparation method thereof. Background Art

[0002] Traumatic bleeding and poor hemostasis are the main causes of patient death during clinical surgery and emergency treatment. When dealing with penetrating wounds such as gunshot wounds and stab wounds, it is impossible to stop bleeding by pressing the bleeding site, so auxiliary hemostatic materials are needed to stop bleeding. The commonly used hemostatic materials in current clinical and emergency treatment include non-woven gauze, commercial gelatin sponges, etc. Non-woven gauze is widely used as the lowest-cost hemostatic material, but it is less effective when dealing with non-compressive hemostatic sites such as penetrating wounds, and it cannot adapt to the repair needs of the wound site and needs to be repeatedly removed, affecting wound healing; although commercial gelatin sponges have a certain shape memory function and can exert a certain amount of pressure on the bleeding site, they still have problems such as slow hemostasis and rapid degradation. During use, blood often fails to wet the sponge in time, resulting in heavy bleeding. Current improvements in this area often involve adding materials like chitosan and silk fibroin to sponges to enhance their hydrophilicity and antibacterial properties. While these materials possess a certain degree of shape memory, they cannot fundamentally address the issue of slow non-compressive hemostasis and also suffer from the mismatch between wound healing and material degradation. Therefore, a material that can meet the demand for rapid, non-compressive hemostasis is urgently needed. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a composite pore structure collagen hemostatic sponge and its preparation method. The hemostatic sponge has excellent unilateral or bilateral bleeding blocking ability and has shape memory function, which solves the problems of existing hemostatic materials in the non-compression hemostasis process such as slow hemostasis speed, degradation and mismatch between wound healing, and has great application value.

[0004] The present invention solves the above technical problems with the following technical solution: a method for preparing a composite pore structure collagen hemostatic sponge is provided, comprising the following steps:

[0005] (1) Dissolve recombinant humanized collagen in ultrapure water to prepare solution B with a concentration of 30-100 mg / mL and solution S with a concentration of 100-200 mg / mL;

[0006] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold according to scheme a or b, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0007] a. Add solution B to freeze first and then add solution S;

[0008] b. Add solution S first and then add solution B, and then add solution S after freezing;

[0009] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an EDC / NHS ethanol solution;

[0010] (4) Soaking the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3) at 0-10°C for 12-36 h, washing and freeze-drying to obtain a composite pore structure collagen hemostatic sponge.

[0011] Furthermore, in step (1), the collagen is at least one of recombinant humanized type I collagen, recombinant humanized type II collagen, recombinant humanized type III collagen, recombinant humanized type XVII collagen, animal-derived type I collagen, animal-derived type II collagen, animal-derived type III collagen, and animal-derived type XVII collagen.

[0012] Furthermore, in step (2), the volume ratio of solution B and solution S in scheme a is 150 μL:120-180 μL.

[0013] Furthermore, in step (2), the volume ratio of solution S, solution B, and solution S in the order of addition in scheme b is 100 μL:80-120 μL:80-120 μL.

[0014] Furthermore, in step (3), the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the EDC / NHS ethanol solution is 30-50 mg / mL, and the concentration of N-hydroxysuccinimide is 5-15 mg / mL.

[0015] Furthermore, in step (3), the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%.

[0016] Furthermore, in step (3), the pH of the EDC / NHS ethanol solution is 5-6.

[0017] Furthermore, in step (4), ultrapure water is used for washing for 12-24 h.

[0018] The present invention also provides a composite pore structure collagen hemostatic sponge prepared by the preparation method of the composite pore structure collagen hemostatic sponge.

[0019] The present invention has the following beneficial effects:

[0020] 1. This study designed two sponge materials with composite pore structures: SB (Scheme a) and SBS (Scheme b), which block unilateral and bilateral bleeding, respectively. Using a directional freezing mold, collagen molecules were arranged in an orderly fashion using ice crystals, and collagen solutions of varying concentrations were added in batches to successfully create these composite pore structures. For material SB, solution B is added first during the directional freezing process. The lower-concentration solution B, due to its fewer collagen molecules, can form larger directional pores, which can rapidly absorb blood during hemostasis through the capillary effect. The subsequent addition of solution S, due to its lower supercooling and higher number of collagen molecules, can form denser, disordered, non-directional pores, which can block and coagulate the absorbed blood. This composite pore structure of directional large pores / non-directional small pores can achieve rapid absorption and hemostasis for unilateral bleeding. In animal experiments, the hemostasis time of this sponge was longer than that of commercial gelatin sponges, and the bleeding volume was reduced by approximately 70% compared with commercial gelatin sponges. For material SBS, the high-concentration solution S added at the beginning and end forms a microporous structure with small pores on the sides and large pores in the middle. This can effectively block bleeding on both sides of the wound in penetrating wounds. In animal experiments, the hemostasis time was significantly longer than that of commercial gelatin sponges, and the bleeding volume was reduced by approximately 50%.

[0021] 2. The sponge of this invention, treated with an EDC / NHS ethanol solution, possesses shape memory properties. This sponge can be compressed in a dry state and then rebound upon contact with body fluids and other liquids, exerting pressure on its surroundings, effectively performing non-compressive hemostasis. Its compression rate can reach 80%, with a recovery time of approximately 1-4 seconds and a volume recovery rate of 90%. This shape memory property surpasses the 20-second recovery time of existing commercial gel sponges and the 5-10-second recovery time of chitosan sponges.

[0022] 3. The sponge of the present invention can significantly affect the degradation performance of the material by regulating the EDC / NHS cross-linking time and the concentration of the collagen solution, thereby adapting to the wound healing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a general view of the collagen hemostatic sponge SB with a repeated pore structure prepared in Example 1;

[0024] Figure 2 This is a microscopic morphology of the composite pore structure collagen hemostatic sponge SB prepared in Example 1;

[0025] Figure 3 The graphs show the cyclic compression mechanical properties test results of each group of sponge materials;

[0026] Figure 4This is a statistical diagram of the shape memory recovery time and recovery rate of the sponge material SB;

[0027] Figure 5 Degradation curves of sponge materials in each group;

[0028] Figure 6 is the swelling curve of each group of sponge materials;

[0029] Figure 7 This is a comparison of the proliferation of L929 cells in each group of sponge materials;

[0030] Figure 8 The staining observation pictures of L929 cells in the sponge materials of each group;

[0031] Figure 9 The diagrams show the general view of hemolysis and the statistical results of hemolysis rate of sponge materials in each group;

[0032] Figure 10 Illustration of the practical operation of hemostasis for tail amputation and liver penetrating wound in SD rats;

[0033] Figure 11 The graphs show the unilateral hemostatic performance test results of the sponge materials in each group;

[0034] Figure 12 The graphs show the test results of bilateral hemostasis performance of penetrating wounds of sponge materials in each group;

[0035] Figure 13 These are the scanning electron microscope observation results of each group of sponge materials. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0037] Example 1

[0038] A composite pore structure collagen hemostatic sponge (SB), the preparation method of which comprises the following steps:

[0039] (1) Recombinant humanized type III collagen was dissolved in ultrapure water to prepare solution B with a concentration of 60 mg / mL and solution S with a concentration of 120 mg / mL, respectively;

[0040] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0041] a. First add 150 μL of solution B to freeze, then add 150 μL of solution S;

[0042] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0043] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 24 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a composite pore structure collagen hemostatic sponge.

[0044] Example 2

[0045] A composite pore structure collagen hemostatic sponge (SBS), the preparation method of which comprises the following steps:

[0046] (1) Recombinant humanized type III collagen was dissolved in ultrapure water to prepare solution B with a concentration of 60 mg / mL and solution S with a concentration of 120 mg / mL, respectively;

[0047] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0048] b. First add 100 μL of solution S and freeze it, then add 100 μL of solution B, and then add 100 μL of solution S again after freezing.

[0049] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0050] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 12 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a composite pore structure collagen hemostatic sponge.

[0051] Example 3

[0052] A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps:

[0053] (1) Recombinant humanized type I collagen, recombinant humanized type II collagen, and recombinant humanized type III collagen were dissolved in ultrapure water to prepare solution B with a concentration of 30 mg / mL and solution S with a concentration of 100 mg / mL, respectively;

[0054] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0055] a. First add 150 μL of solution B to the ice tube, then add 120 μL of solution S.

[0056] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5, the EDC concentration is 30 mg / mL, and the NHS concentration is 5 mg / mL;

[0057] (4) The precursor sponge obtained in step (2) was immersed in the EDC / NHS ethanol solution obtained in step (3), cross-linked at 0°C for 36 h, washed with ultrapure water for 24 h, and freeze-dried to obtain a composite pore structure collagen hemostatic sponge.

[0058] Example 4

[0059] A recombinant collagen hemostatic sponge, the preparation method of which comprises the following steps:

[0060] (1) Recombinant humanized type I collagen was dissolved in ultrapure water to prepare solution B with a concentration of 100 mg / mL and solution S with a concentration of 200 mg / mL;

[0061] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0062] b. First add 100 μL of solution S and freeze it, then add 120 μL of solution B, and then add 120 μL of solution S again after freezing.

[0063] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 6, the EDC concentration is 50 mg / mL, and the NHS concentration is 15 mg / mL;

[0064] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 10°C for 30 h, wash with ultrapure water for 20 h, and freeze-dry to obtain a recombinant collagen hemostatic sponge.

[0065] Example 5

[0066] A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps:

[0067] (1) Dissolve animal-derived type II collagen in ultrapure water to prepare solution B with a concentration of 60 mg / mL and solution S with a concentration of 120 mg / mL;

[0068] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0069] a. First add 150 μL of solution B to freeze, then add 150 μL of solution S;

[0070] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0071] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 24 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a composite pore structure collagen hemostatic sponge.

[0072] Example 6

[0073] A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps:

[0074] (1) Dissolve animal-derived type I collagen in ultrapure water to prepare solution B with a concentration of 60 mg / mL and solution S with a concentration of 120 mg / mL;

[0075] (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directionally freezing, and freeze-drying to obtain a precursor sponge;

[0076] b. First add 100 μL of solution S and freeze it, then add 100 μL of solution B, and then add 100 μL of solution S again after freezing.

[0077] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0078] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 12 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a composite pore structure collagen hemostatic sponge.

[0079] Comparative Example 1

[0080] A recombinant collagen sponge (S), the preparation method of which comprises the following steps:

[0081] (1) Dissolve recombinant humanized type III collagen in ultrapure water to prepare a solution S with a concentration of 120 mg / mL;

[0082] (2) Add 300 μL of the solution S obtained in step (1) into a pre-cooled mold for directional freezing and freeze-drying to obtain a precursor sponge;

[0083] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0084] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 24 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a recombinant collagen sponge.

[0085] Comparative Example 2

[0086] A recombinant collagen sponge (B), the preparation method of which comprises the following steps:

[0087] (1) Dissolve recombinant humanized type III collagen in ultrapure water to prepare solution B with a concentration of 60 mg / mL;

[0088] (2) Add 300 μL of solution B obtained in step (1) into a pre-cooled mold for directional freezing and freeze-drying to obtain a precursor sponge;

[0089] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to MES ethanol buffer solution to obtain EDC / NHS ethanol solution; the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%, the pH is 5.5, the EDC concentration is 40 mg / mL, and the NHS concentration is 10 mg / mL;

[0090] (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 4°C for 24 h, wash with ultrapure water for 12 h, and freeze-dry to obtain a recombinant collagen sponge.

[0091] Test Example 1

[0092] (1) The general view of the composite porous structure collagen hemostatic sponge (SB) prepared in Example 1 is as follows Figure 1 As shown, the composite pore structure collagen hemostatic sponge is a white block structure with excellent compressibility.

[0093] (2) The microscopic morphology of the composite porous structure collagen hemostatic sponge (SB) prepared in Example 1 is as follows: Figure 2 As shown, the interior of the recombinant collagen hemostatic sponge is a porous transport structure, with a composite pore structure of oriented large pores and disordered small pores, and clear interfaces between the composite pores.

[0094] (3) The cyclic compression mechanical properties of the sponges (SB, SBS, S and B) prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 were tested using a universal mechanical testing machine (EZ-SX, Shimadzu). The results are as follows: Figure 3 shown.

[0095] Depend on Figure 3 It can be seen that materials in different groups have certain cyclic compression performance and shape memory function. The mechanical properties of sponge in dry state (0) and wet state (1-5) are quite different, but its mechanical properties only change slightly under multiple cyclic compression.

[0096] During the test, PBS solution was added dropwise after compression to allow the sponge material SB to absorb liquid and recover the memorized shape. The shape memory recovery time and recovery rate statistics of the sponge material SB prepared in Example 1 are shown in the figure below: Figure 4 shown.

[0097] Depend on Figure 4 It can be seen that sponge SB can recover its shape within 1-3 s, and its shape recovery rate is as high as over 95%, which has extremely excellent shape memory function.

[0098] Test Example 2

[0099] In order to analyze the degradation performance of sponges, sponges SB, SBS, S and B were immersed in 1 mL of 50 U / mL collagenase solution (dissolved in PBS) and placed in a constant temperature shaker at 37°C and oscillated at 100 rpm. After soaking for a period of time, they were taken out, freeze-dried and weighed until degradation equilibrium was reached. The degradation curves are shown in Figure 2. Figure 5 shown.

[0100] Depend on Figure 5 It can be seen that sponge materials with different micropore structures exhibit different degradation properties. In particular, the S sponge with a small pore structure completely degraded within 24 hours. This can be attributed to its smaller pore structure hindering the entry of the EDC / NHS crosslinking solution, resulting in a low degree of crosslinking and rapid degradation. The different degradation rates of different structures provide the possibility of adapting to the wound healing environment.

[0101] Test Example 3

[0102] In order to analyze the swelling characteristics of sponges, sponges SB, SBS, S and B were immersed in PBS solution, placed in a constant temperature shaker, and shaken at 100 rpm at 37 °C. After soaking for a period of time, the material was taken out and weighed until swelling equilibrium was reached. The swelling curve is shown in Figure 2. Figure 6 shown.

[0103] Depend on Figure 6 As can be seen, the more S solution added to the sponge material, the lower the swelling rate. This is because the sponge prepared with a higher concentration of recombinant humanized collagen solution has a smaller pore structure. SBS, on the other hand, has a faster swelling equilibrium time, which is due to the faster liquid absorption rate achieved by the microscopic pore structure design of the SBS sponge.

[0104] Test Example 4

[0105] In order to analyze the biocompatibility of the sponge, stably passaged L929 cells were taken and digested from the culture dish using trypsin. The digestion was then terminated using 10% serum α-MEM medium and resuspended for counting. α-MEM medium (Hyclone, USA) was supplemented with 1% penicillin / streptomycin (PS, Gibco). Subsequently, 2000 resuspended L929 cells were added to each well of a 24-well plate, and the plate was cultured at 5% CO2 and 37°C for 1 day. The biocompatibility of the recombinant humanized collagen sponge was characterized by two-dimensional cell culture. Sponges S, B, SB, and SBS were taken, sterilized by UV, and then placed in the above-mentioned 24-well plate seeded with L929 cells, one per well, and a blank control group was set up. Subsequently, 1.5 mL of the above-mentioned medium containing 10% serum was added to each well, and the samples were cultured under the same conditions. CCK-8 (Dojindo, Japan) was used to detect cell proliferation in different sponge materials at 1 day, 3 days, and 7 days, respectively. The results are shown in the figure. Figure 7 The results showed that the cell activity gradually increased over time, and there was no significant difference between the groups, indicating that the material had no obvious cytotoxicity.

[0106] The cell proliferation and morphology after FDA / PI and DAPI staining were observed under an inverted fluorescence microscope. Figure 8 As shown in the results, there was no significant difference in the morphology and number of cells between the sponge materials in each group and the blank control group, which indicated that the materials did not hinder the proliferation and spreading of cells.

[0107] Test Example 5

[0108] The blood compatibility of the sponge was analyzed to evaluate its feasibility in hemostasis scenarios. A certain amount of venous blood was collected from New Zealand white rabbits using an anticoagulant blood collection tube using the ear vein blood collection technique. The venous blood was washed with PBS solution, centrifuged, and the lower layer of red blood cells was removed. The washing process was repeated three times. Then UP water was added to the removed red blood cell solution to promote hemolysis to form a positive control group (+). PBS solution was added to the removed red blood cell solution and S, B, SB and SBS were added as the experimental group. PBS solution was added to the removed red blood cell solution as a negative control group (-). The solution was then placed on a 37°C shaker for 1 hour, centrifuged and photographed. 200 μl of the supernatant after centrifugation was taken out and added to a 96-well plate. The absorbance at 562 nm was measured using an enzyme reader, and the absorbance data was processed to obtain the hemolysis rate. The gross hemolysis image and hemolysis rate statistical results were obtained as shown below. Figure 9 shown.

[0109] Depend on Figure 9It can be seen that the hemolysis rate of each group was at a low level, all below 1.5%, and the hemolysis rate of the SB group was slightly higher than that of other groups. This is because the SB sponge has exposed directional macropores, and blood cells are easily absorbed and squeezed under the action of capillary force, causing slight rupture.

[0110] Test Example 6

[0111] The non-compressive hemostatic performance of the sponge of the present invention and the existing commercial gelatin sponge was compared. Two models of hemostasis were selected: tail hemostasis in SD rats and liver penetrating wound hemostasis in SD rats (see the actual operation diagram). Figure 10 unilateral and bilateral bleeding, respectively. All surgical instruments were sterilized under high temperature and high pressure.

[0112] (1) After SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg / mL), the tails of the SD rats were cut off 8 cm from the base using surgical instruments. After the blood flow from the tail cut was stable, the sponge materials of each group were quickly deformed and placed at the bleeding site. The bleeding time and amount were recorded to obtain the hemostatic performance data of the sponge. The data processing results are shown in the figure. Figure 11 shown.

[0113] Depend on Figure 11 It can be seen that in the unilateral bleeding model, in terms of bleeding volume, the four recombinant humanized collagen sponge groups are superior to the existing commercial gelatin sponges, and the S sponge has a larger bleeding volume. This is because its pore size is small, and most of the blood flows out from the surface of the sponge material without infiltrating the sponge, which is similar to the actual situation of commercial gelatin sponges; in terms of hemostasis time, the SB sponge has the shortest hemostasis time, about 40-45 s, and is better than the 45-55 s of the commercial gelatin sponge, which is due to the coordinated hemostasis effect of its microscopic composite pores; the B sponge shows the longest hemostasis time, about 80-110 s, which is because the pore size of its internal directional microchannels is large, and the blood cannot form effective blocking coagulation inside it after rapid infiltration.

[0114] (2) After SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg / mL), the thorax of the SD rats was opened along the lower edge of the thoracic vertebrae using surgical instruments. The rat liver was pulled out using sterile blunt forceps and placed on a plastic board. A 10 mm punch was used to punch a through wound on the liver. The sponge materials of each group were quickly deformed and placed on the bleeding site. The bleeding time and amount were recorded to obtain the hemostatic performance data of the recombinant humanized collagen sponge. The data processing results are shown in Figure 2. Figure 12 shown.

[0115] Depend on Figure 12It can be seen that under the penetrating wound bleeding model, each group of composite pore structure collagen hemostatic sponges are superior to commercial gelatin sponges in terms of hemostasis time and bleeding volume, and the hemostasis time is shortened by 40-80 s, which is due to the internal compression brought by its excellent shape memory function; among the collagen hemostatic sponges, the SBS sponge shows the fastest hemostasis time, about 40-55 s, which is better than the 90-150 s of the commercial gelatin sponge. This is due to the dense microporous structure design of the SBS sponge on both sides and sparse inside and its shape memory function. The shape memory function of the sponge and its rapid recovery speed can enable it to quickly fill the penetrating wound site tightly, and the design of the composite microporous structure can enable the blood in the penetrating wound site to quickly enter the sponge and form a blockage in the tight pore structure on both sides, thereby quickly stopping bleeding.

[0116] (3) After the tail was cut to stop bleeding, the material was removed and fixed with glutaraldehyde for 168 h. It was then dehydrated with ethanol gradient (30%, 50%, 70%, 90% and 100%), and then critical point dried. Finally, it was observed using a scanning electron microscope to determine the movement and residence pattern of blood cells inside the material. The results are as follows: Figure 13 As shown, a, b and c are micrographs of the middle part of the materials after hemostasis of S sponge, B sponge and SB sponge, respectively; c1, c2 and c3 are microscopic morphologies of the small pore surface, oriented large pore surface and high-magnification photographs of the platelets in the activated state inside the SB sponge, respectively.

[0117] Depend on Figure 13 As can be seen, images A to C show only a small number of red blood cells entering the S sponge, while the B sponge is heavily covered with red blood cells, with no obvious signs of platelet coagulation. In the SB sponge, red blood cells clog the directional channels at the ends, forming a large network. Images C1-C3 show no red blood cell exudation from the small pores, while a large number of red blood cells enter along the directional channels of the large pores, revealing a clear network of activated platelets. Comparison of the micrographs confirms the effectiveness of the sponge's internal pore structure design.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a composite pore structure collagen hemostatic sponge, characterized in that: The following steps are involved: (1) Dissolve collagen in ultrapure water to prepare solution B with a concentration of 30-100 mg / mL and solution S with a concentration of 100-200 mg / mL; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold according to scheme a, directionally freezing, and freeze-drying to obtain a precursor sponge; a. Add solution B to freeze first and then add solution S; the volume ratio of solution B and solution S is 150 μL: 120-180 μL; (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the MES ethanol buffer solution to obtain EDC / NHS ethanol solution; (4) Soaking the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3) at 0-10°C for 12-36 h, washing and freeze-drying to obtain a composite pore structure collagen hemostatic sponge.

2. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, wherein: In step (1), the collagen is at least one of recombinant humanized type I collagen, recombinant humanized type II collagen, recombinant humanized type III collagen, recombinant humanized type XVII collagen, animal-derived type I collagen, animal-derived type II collagen, animal-derived type III collagen, and animal-derived type XVII collagen.

3. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, wherein: In step (3), the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the EDC / NHS ethanol solution is 30-50 mg / mL, and the concentration of N-hydroxysuccinimide is 5-15 mg / mL.

4. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, wherein: In step (3), the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%.

5. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, wherein: In step (3), the pH of the EDC / NHS ethanol solution is 5-6.

6. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, wherein: In step (4), wash with ultrapure water for 12-24 h.

7. A composite pore structure collagen hemostatic sponge prepared by the method for preparing the composite pore structure collagen hemostatic sponge according to any one of claims 1 to 6.

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