Collagen hemostatic sponge with composite pore structure and preparation method of collagen hemostatic sponge

Abstract

The invention discloses a collagen hemostatic sponge with a composite pore structure and a preparation method thereof, and relates to the technical field of biomedical materials.The preparation method comprises the following steps that 1, collagen is dissolved in ultrapure water, and a solution B and a solution S with different concentrations are prepared respectively; (2) adding the solution B and the solution S into a mold according to different schemes, performing directional freezing, and performing freeze drying to obtain a precursor sponge; (3) adding EDC and NHS into the MES ethanol buffer solution to obtain an EDC / NHS ethanol solution; and (4) soaking the precursor sponge in an EDC / NHS ethanol solution for cross-linking, washing and freeze-drying to obtain the collagen hemostatic sponge with the composite pore structure. The hemostatic sponge has excellent single-side or double-side bleeding blocking capacity, has a shape memory function, solves the problems that an existing hemostatic material is too slow in hemostatic speed, not matched with wound healing in degradation and the like in the non-compression hemostasis process, and has high application value.

Description

Technical Field

[0001] The 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 existing 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 form a certain pressure on the bleeding site, they still have problems such as slow hemostasis and too fast degradation. During use, blood often fails to wet the sponge in time, resulting in heavy bleeding. At present, the improvement in this field often adds materials such as chitosan and silk fibroin to increase the hydrophilicity and antibacterial properties of the sponge. Although the prepared materials have a certain shape memory function, they cannot fundamentally solve the problem of slow non-compression hemostasis, and there is also the problem of mismatch between wound healing and material degradation. Therefore, there is an urgent need for a material that can meet the demand for rapid non-compression hemostasis. 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 a preparation method thereof, which has excellent unilateral or bilateral bleeding blocking ability and shape memory function, solves the problems of slow hemostasis speed, mismatch between degradation and wound healing, etc. of existing hemostatic materials in the non-compression hemostasis process, and has great application value. The technical solution of the present invention to solve the above technical problem is as follows: a method for preparing a composite pore structure collagen hemostatic sponge is provided, comprising the following steps: (1) Dissolving 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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold according to scheme a or b, directional freezing, and freeze drying to obtain a precursor sponge; a. First add solution B to freeze and then add solution S; b. First add solution S to freeze, then add solution B, and then add solution S after freezing; (3) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an EDC / NHS ethanol solution; (4) Soaking the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-linking at 0-10°C for 12-36 h, washing and freeze-drying to obtain a composite pore structure collagen hemostatic sponge.

[0004] 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.

[0005] Furthermore, in step (2), the volume ratio of solution B and solution S in scheme a is 150 μL:120-180 μL.

[0006] Furthermore, in step (2), in scheme b, the volume ratio of solution S, solution B and solution S in the order of addition is 100 μL: 80-120 μL: 80-120 μL.

[0007] 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.

[0008] Furthermore, in step (3), the ethanol concentration in the EDC / NHS ethanol solution is not less than 90%.

[0009] Furthermore, in step (3), the pH of the EDC / NHS ethanol solution is 5-6.

[0010] Furthermore, in step (4), ultrapure water is used for washing for 12-24 h.

[0011] 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.

[0012] The present invention has the following beneficial effects: 1. The present invention designs two sponge materials with composite pore structures, namely SB (Scheme a) and SBS (Scheme b) sponges, which have the functions of unilateral and bilateral bleeding blocking, respectively. Using a directional freezing mold, ice crystals are used to arrange collagen molecules in an orderly manner, and collagen solutions of different concentrations are added in batches to successfully prepare the above-designed composite pore structure. For material SB, since solution B is added first during the directional freezing process, the lower concentration solution B can form larger directional channels due to its less collagen molecules, and can use the capillary effect to achieve rapid absorption of blood during the hemostasis process. The solution S added later can form denser disordered non-directional channels due to its lower supercooling and more collagen molecules, which can achieve the blocking and coagulation effect on 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 is better than that of commercial gelatin sponge, and the amount of bleeding is reduced by about 70% compared with that of commercial gelatin sponge. For material SBS, due to the addition of high-concentration solution S before and after, it forms a microporous structure with small pores on both sides and large pores in the middle. It can effectively block bleeding on both sides at the same time in penetrating injuries. In animal experiments, the hemostasis time is significantly better than that of commercial gelatin sponge, and the amount of bleeding is reduced by about 50% compared with that of commercial gelatin sponge.

[0013] 2. The sponge of the present invention has shape memory function due to the treatment of EDC / NHS ethanol solution. The sponge can be compressed in a dry state, and then rebound and exert pressure on the surrounding after contacting body fluids and other liquids, and can effectively play a role in non-compressive hemostasis scenarios. Its compression rate can reach 80%, the recovery time is about 1-4 s, and the volume recovery rate is 90%. The shape memory function is better than the 20 s recovery time of existing commercial gel sponges and the 5-10 s recovery time of chitosan sponges.

[0014] 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

[0015] Figure 1 This is a general view of the collagen hemostatic sponge SB with a repeated pore structure prepared in Example 1; Figure 2 This is a microscopic morphology of the composite pore structure collagen hemostatic sponge SB prepared in Example 1; Figure 3 The graphs are the test results of cyclic compression mechanical properties of each group of sponge materials; Figure 4 It is a statistical diagram of shape memory recovery time and recovery rate of sponge material SB; Figure 5It is the degradation curve diagram of each group of sponge materials; Figure 6 It is the swelling curve diagram of each group of sponge materials; Figure 7 This is a comparison of the proliferation of L929 cells in each group of sponge materials; Figure 8 The staining observation pictures of L929 cells in each group of sponge materials; Fig. 9 The general view of hemolysis and the statistical result of hemolysis rate of each group of sponge materials are shown in the figure; Fig.10 Practical diagram of hemostasis of tail amputation and liver penetration wound in SD rats; Fig.11 The graphs are the test results of unilateral hemostasis performance of sponge materials in each group; Fig.12 The graphs show the test results of bilateral hemostasis performance of penetrating wounds of sponge materials in each group; Fig.13 These are the scanning electron microscope observation results of each group of sponge materials. DETAILED DESCRIPTION

[0016] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0017] Example 1 A composite pore structure collagen hemostatic sponge (SB), the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type III 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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a for directional freezing and freeze drying to obtain a precursor sponge; a. First add 150 μL of solution B to freeze, then add 150 μL of solution S; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0018] Example 2 A composite pore structure collagen hemostatic sponge (SBS), the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type III 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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directional freezing, and freeze drying to obtain a precursor sponge; 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 after freezing; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0019] Example 3 A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type I collagen, recombinant humanized type II collagen, and recombinant humanized type III collagen 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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a for directional freezing and freeze drying to obtain a precursor sponge; a. First add 150 μL of solution B and freeze it, then add 120 μL of solution S; (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; 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; (4) Soak the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-link at 0°C for 36 h, wash with ultrapure water for 24 h, and freeze-dry to obtain a composite pore structure collagen hemostatic sponge.

[0020] Example 4 A recombinant collagen hemostatic sponge, the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type I collagen in ultrapure water to prepare solution B with a concentration of 100 mg / mL and solution S with a concentration of 200 mg / mL; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directional freezing, and freeze drying to obtain a precursor sponge; 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 after freezing; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0021] Example 5 A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps: (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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme a for directional freezing and freeze drying to obtain a precursor sponge; a. First add 150 μL of solution B to freeze, then add 150 μL of solution S; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0022] Example 6 A composite pore structure collagen hemostatic sponge, the preparation method of which comprises the following steps: (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; (2) adding the solution B and solution S obtained in step (1) into a pre-cooled mold in the order of scheme b, directional freezing, and freeze drying to obtain a precursor sponge; 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 after freezing; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0023] Comparative Example 1 A recombinant collagen sponge (S), the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type III collagen in ultrapure water to prepare a solution S with a concentration of 120 mg / mL; (2) adding 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; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0024] Comparative Example 2 A recombinant collagen sponge (B), the preparation method of which comprises the following steps: (1) Dissolve recombinant humanized type III collagen in ultrapure water to prepare solution B with a concentration of 60 mg / mL; (2) adding 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; (3) Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the MES ethanol buffer solution to obtain an 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; (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.

[0025] Test Example 1 (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.

[0026] (2) The microscopic morphology of the composite pore 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, and a composite pore structure of oriented large pores and disordered small pores can be seen, and there is a clear interface between the composite pores.

[0027] (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.

[0028] 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.

[0029] 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.

[0030] Depend on Figure 4 It can be seen that sponge SB can restore its shape within 1-3 s, and its shape recovery rate is as high as over 95%, which has extremely excellent shape memory function.

[0031] Test Example 2 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 the degradation equilibrium was reached. The degradation curves are shown in Figure 2. Figure 5 shown.

[0032] Depend on Figure 5 It can be seen that under different microscopic pore structures, sponge materials show different degradation properties, especially S sponge with small pore structure, which is completely degraded in 24 hours. This can be attributed to its smaller pore structure that hinders the entry of EDC / NHS cross-linking liquid, resulting in low cross-linking degree and rapid degradation. Different degradation rates of different structures provide the possibility of adapting to the wound healing environment.

[0033] Test Example 3 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 curves are shown in Figure 6 shown.

[0034] Depend on Figure 6 It can be seen that the more S solution is added to the sponge material, the lower the swelling rate is, because the sponge prepared by the higher concentration of recombinant humanized collagen solution has a smaller pore structure. SBS has a faster swelling equilibrium time, which is due to the faster liquid absorption rate brought by the microscopic pore structure design of the SBS sponge.

[0035] Test Example 4 In order to analyze the biocompatibility of the sponge, the stable passaged L929 cells were taken and digested from the culture dish with trypsin, and then the digestion was terminated with α-MEM medium containing 10% serum and resuspended for counting. 1% penicillin / streptomycin (PS, Gibco) was added to the α-MEM medium (Hyclone, USA). Then, a 24-well plate was taken, 2000 resuspended L929 cells were added to each well, and the well 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 ultraviolet and then placed in the above-mentioned 24-well plate inoculated with L929 cells, 1 per well, and a blank control group was set up. Subsequently, 1.5 mL of the above-mentioned culture 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 following table. Figure 7 The results showed that over time, the cell activity gradually increased, and there was no obvious gap between the groups, indicating that the material had no obvious cytotoxicity.

[0036] Inverted fluorescence microscopy was used to observe cell proliferation and morphology after FDA / PI and DAPI staining. Figure 8 As shown, the results showed that 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 indicates that the material did not hinder the proliferation and spreading of cells.

[0037] Test Example 5 The blood compatibility of the sponge was analyzed to evaluate its feasibility in hemostasis scenarios. Some 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, and PBS solution was added to the removed red blood cell solution as a negative control group (-). The solution was then placed in a 37°C shaker for 1 h, centrifuged and photographed, and 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 the statistical results of the hemolysis rate were obtained as shown in the following figure. Fig. 9 shown.

[0038] Depend on Fig. 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.

[0039] Test Example 6 The non-compressive hemostatic performance of the sponge of the present invention and the existing commercial gelatin sponge was compared and studied. Two models of SD rat tail hemostasis and SD rat liver penetrating wound hemostasis were selected (the actual operation diagram is as follows Fig.10 ), corresponding to unilateral and bilateral bleeding, respectively. All surgical instruments were sterilized with high temperature and high pressure.

[0040] (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 root 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. Fig.11 shown.

[0041] Depend on Fig.11 It can be seen that under the unilateral bleeding model, in terms of bleeding volume, the four recombinant humanized collagen sponge groups are better than 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 commercial gelatin sponges, 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.

[0042] (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 make a penetrating wound on the liver. The sponge materials of each group were quickly deformed and placed at the bleeding location. 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. Fig.12 shown.

[0043] Depend on Fig.12It can be seen that under the penetrating injury 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 injury site tightly, and the design of the composite microporous structure can enable the blood at the penetrating injury site to quickly enter the sponge and form a blockage in the tight pore structure on both sides, thereby quickly stopping bleeding.

[0044] (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 gradient ethanol (30%, 50%, 70%, 90% and 100%) and critical point dried. Finally, it was observed with a scanning electron microscope to determine the movement and residence pattern of blood cells inside the material. The results are as follows: Fig.13 As shown, a, b and c are micrographs of the middle parts of the S sponge, B sponge and SB sponge materials after hemostasis, respectively, and c1, c2 and c3 are micrographs of the small pore surface of the SB sponge, the oriented large pore surface and high-magnification photographs of the platelets in the internal activated state, respectively.

[0045] Depend on Fig.13 It can be seen that only a small number of red blood cells enter the S sponge, and a large number of red blood cells are covered inside the B sponge. There is no obvious sign of platelet coagulation. The red blood cells inside the SB sponge are blocked at the end of the directional channel and form a large number of reticular tissues. In the figure, c1-c3 shows that there is no red blood cell exudation on the small hole surface, and a large number of red blood cells enter along the directional channel on the large hole surface. There are clear activated platelets connected into a network inside. The comparison of microscopic photos confirms the effectiveness of the internal pore structure design of the sponge material.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope 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) Dissolving 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 or b, directional freezing, and freeze drying to obtain a precursor sponge; a. First add solution B to freeze and then add solution S; b. First add solution S to freeze, then add solution B, and then add solution S after freezing; (3) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the MES ethanol buffer solution to obtain an EDC / NHS ethanol solution; (4) Soaking the precursor sponge obtained in step (2) in the EDC / NHS ethanol solution obtained in step (3), cross-linking 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, characterized in that: 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, characterized in that: In step (2), the volume ratio of solution B and solution S in scheme a is 150 μL:120-180 μL.

4. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, characterized in that: 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.

5. The method for preparing the composite pore structure collagen hemostatic sponge according to claim 1, characterized in that: 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.

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

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

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

9. A composite pore structure collagen hemostatic sponge obtained by the method for preparing the composite pore structure collagen hemostatic sponge according to any one of claims 1 to 8.

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