An absorbable arterial hemostatic sponge, its preparation method and application

Abstract

This invention discloses an absorbable arterial hemostatic sponge, its preparation method, and its applications. The hemostatic sponge is prepared by a cross-linking reaction and freeze-drying of γ-polyglutamic acid, gelatin, carboxymethyl chitosan, collagen, glycerol, and a cross-linking agent system. The cross-linking agent system includes a water-soluble carbodiimide as the main cross-linking agent, ethylene glycol diglycidyl ether as the first auxiliary cross-linking agent, and ammonium chloride as the second auxiliary cross-linking agent. This hemostatic sponge has a uniform microporous structure, an absorption rate of over 80 g / g in 30 minutes, excellent expansion performance, good biocompatibility, and a mild preparation process that does not require strong acids or alkalis, making it easy to scale up for industrial production. The sponge of this invention combines the advantages of high expansion, absorbability, mechanical flexibility, and simple processing, making it suitable for hemostasis in clinical surgery and trauma emergency care.

Description

Technical Field

[0001] This invention relates to the field of medical hemostatic materials technology, specifically to an absorbable arterial hemostatic sponge, its preparation method, and its application. Background Technology

[0002] In clinical surgery, traffic accidents, war, and natural disasters, uncontrolled bleeding caused by trauma is a major cause of shock and even death in wounded soldiers. In surgical procedures, especially when operating on richly vascularized solid organs such as the liver, spleen, and nasal cavity, widespread bleeding and oozing are unavoidable challenges. Therefore, developing rapid, effective, biocompatible hemostatic materials with minimal side effects has significant clinical implications and a broad market demand.

[0003] Local packing is one of the most commonly used hemostatic methods in clinical practice. Its advantages lie in its good compression protection of the bleeding site, its definite hemostatic effect, its long duration of action, and its promotion of wound healing. Ideal packing materials should have good elasticity, conformability, and trimmability to adapt to the packing requirements of different wound surfaces. At the same time, they should ideally also have absorbability, anti-inflammatory, swelling-reducing, and wound-healing properties.

[0004] Currently, commercially available expandable hemostatic sponges are mainly made of polyvinyl alcohol acetal. This type of material is highly hydrophilic, capable of absorbing 20 times its own weight in liquid and expanding 10 times, exhibiting a flexible texture and good hemostatic effect. However, this type of material has the following prominent drawbacks: ① Due to the limitations of the polyvinyl alcohol (CC) main chain, this type of sponge is non-degradable in vivo and can only be used externally or as a short-term packing material. Removal can easily cause secondary trauma and severe pain, and the disposal of waste materials as medical waste is costly and poses potential environmental hazards. ② Clinical use shows that its hemostatic effect is poor for bleeding from pulsatile arteries or patients with coagulation disorders; its water absorption and coagulation-promoting abilities need further improvement. ③ The material itself has no antibacterial properties; direct packing can easily lead to wound infection or even suppuration. Clinical use requires additional application of antibiotics, which is cumbersome, results in uneven drug distribution, and has poor sustained-release effects.

[0005] Other absorbable hemostatic materials currently in use also have their limitations. For example, microfibrillated collagen derived from bovine dermis poses a risk of allergic reactions and increased infection; calcium alginate dressings, while biocompatible and hydrophilic, liquefy rapidly upon contact with water, lacking sufficient toughness and elasticity, resulting in insufficient pressure on the wound and difficulty in cleaning; gelatin sponges, although biocompatible, are not ideal for hemostasis when coagulation mechanisms are impaired, residues easily form adhesions, and they also lack antibacterial properties; oxidized-carboxymethyl cellulose hemostatic fabrics dissolve rapidly upon contact with blood, lack expansion and compressive force, and are ineffective for significant bleeding.

[0006] Therefore, there is an urgent clinical need to develop a new type of packing hemostatic sponge to overcome the many shortcomings of existing products. Summary of the Invention

[0007] To improve the effectiveness and safety of local tamponade hemostasis, this invention provides an absorbable arterial hemostatic sponge, its preparation method, and its application.

[0008] The first technical solution adopted in this invention is as follows: an absorbable arterial hemostatic sponge, prepared from raw materials containing the following components through a cross-linking reaction and freeze-drying: γ-polyglutamic acid; gelatin; carboxymethyl chitosan; collagen; glycerol; and a cross-linking agent system; wherein the cross-linking agent system includes a main cross-linking agent, a first auxiliary cross-linking agent, and a second auxiliary cross-linking agent; the main cross-linking agent is water-soluble carbodiimide, the first auxiliary cross-linking agent is a glycidyl ether cross-linking agent that can cross-link with gelatin, and the second auxiliary cross-linking agent is an auxiliary agent that can promote the cross-linking of water-soluble carbodiimide with γ-polyglutamic acid.

[0009] Preferably, the first auxiliary crosslinking agent is ethylene glycol diglycidyl ether, and the second auxiliary crosslinking agent is ammonium chloride.

[0010] Preferably, the raw material ratios satisfy at least one of the following: the mass ratio of the main crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.02:1; the mass ratio of the first auxiliary crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.01:1; the mass ratio of the second auxiliary crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.005:1; the mass concentration of γ-polyglutamic acid in the reaction solution is 4.8%-5.1%; the mass ratio of glycerol to the sum of the masses of γ-polyglutamic acid and gelatin is 0.13:1; the mass ratio of gelatin to γ-polyglutamic acid is 0.2:1; the mass ratio of carboxymethyl chitosan to γ-polyglutamic acid is 0.06:1; and the mass ratio of collagen to γ-polyglutamic acid is 0.01:1-0.05:1.

[0011] Preferably, the total mass of the first auxiliary crosslinking agent and the second auxiliary crosslinking agent accounts for 40%-60% of the main crosslinking agent, and the mass ratio of the first auxiliary crosslinking agent to the second auxiliary crosslinking agent is 0.8:1-1.2:1.

[0012] Preferably, the molecular weight of γ-polyglutamic acid is ≥1000kDa.

[0013] Preferably, the hemostatic sponge has a uniform microporous structure with a porosity of 5%-48% and a pore size range of 4μm-170μm.

[0014] Preferably, the hemostatic sponge is degradable in a protease-containing buffer or bacterial culture medium, with a degradation rate of ≥55% after 6 days and ≥50% after 12 days in bacterial culture medium.

[0015] The second technical solution adopted in this invention is as follows: A method for preparing an absorbable arterial hemostatic sponge, characterized by comprising the following steps: Step S1. Dissolve γ-polyglutamic acid, gelatin, carboxymethyl chitosan and glycerol in water and stir until homogeneous to obtain a polymer solution; Step S2. Add the first auxiliary crosslinking agent and the second auxiliary crosslinking agent to the obtained polymer solution to carry out the first crosslinking reaction; Step S3. Then add the main crosslinking agent to carry out the second crosslinking reaction and form a gel; Step S4. Pre-freeze the obtained gel to form a solid shape, and then use freeze-drying method to vacuum dry it to obtain a hemostatic sponge.

[0016] Preferably, the water temperature in step S1 is 35℃-40℃; the total crosslinking reaction time in steps S2 and S3 is 30-60 minutes.

[0017] The third technical solution adopted in this invention is as follows: an absorbable arterial hemostatic sponge, used in the preparation of medical dressings or hemostatic materials for packing and stopping bleeding in clinical surgery and trauma emergency care.

[0018] The present invention has the following beneficial effects: 1. Water Absorption and Swelling Properties and Hemostatic Effect: The hemostatic sponge can absorb more than 80 g / g of water in deionized water within 30 minutes. This high absorbency gives the sponge the ability to quickly absorb blood and exudate from the wound. Through volume expansion, it generates uniform and effective physical pressure on the bleeding site, making it suitable for pulsatile arterial bleeding and large-area oozing. Simultaneously, the collagen in the formula has good tissue adhesion and platelet aggregation promoting effects, allowing platelets to adhere to and aggregate on the bleeding wound to form a blood clot, further enhancing the hemostatic effect. Compared with existing non-absorbable expandable sponges based on polyvinyl alcohol, the sponge of this invention maintains comparable swelling properties while also being absorbable; compared with calcium alginate dressings, it has superior toughness and swelling pressure; and compared with simple collagen-based hemostatic materials, it has stronger erosion resistance and is less likely to be washed away by active bleeding.

[0019] 2. Biodegradability and Biocompatibility: The hemostatic sponge uses γ-polyglutamic acid and collagen as its core raw materials. γ-polyglutamic acid is a polyamino acid synthesized by microbial fermentation, which can be degraded into non-toxic glutamic acid monomers in vivo; collagen is the main structural protein of the extracellular matrix and can be degraded into amino acids by collagenases in vivo. The degradation rate can be controlled by the amount of cross-linking agent and gelatin content to meet the material retention time requirements of different clinical scenarios. The degradation products are all endogenous substances or non-toxic small molecules, with excellent biocompatibility, avoiding secondary damage to wounds and the risk of foreign body residue.

[0020] 3. Microporous Structure: It possesses a uniform three-dimensional porous network structure with a porosity of 5%-48% and a pore size ranging from 4μm to 170μm. By adjusting process parameters such as the crosslinking agent ratio, reaction temperature, reaction time, and gelatin addition, the pore structure can be precisely controlled to adapt to the varying needs of different wounds in terms of material density, water absorption rate, and mechanical strength. The addition of collagen helps improve the toughness and structural integrity of the sponge's pore walls, enabling the sponge to maintain a stable network morphology even after absorbing water and swelling.

[0021] 4. Mechanical Flexibility and Adaptability: This invention significantly improves the sponge's brittleness by adding glycerin as a plasticizer, combined with the natural flexibility of collagen, giving it both softness and elasticity. The sponge can be cut and shaped to fit the wound surface, adhering closely during packing, causing minimal irritation to mucous membranes and tissues, resulting in high patient comfort. Compared to existing gelatin sponges, it provides greater expansion and compression; compared to calcium alginate dressings, it offers superior toughness and elasticity.

[0022] 5. Mild Preparation Process: This invention utilizes a WSC / ethylene glycol diglycidyl ether / ammonium chloride ternary crosslinking system, achieving gelation reaction under mild conditions of 37℃ and 40 minutes, without the need for strong acids, strong alkalis, or high temperature and pressure. This process has been successfully scaled up in a 20L reactor, producing sponges with a uniform white appearance and stable performance, verifying its good scalability and industrialization prospects. Compared with existing polyglutamic acid crosslinking technologies, this process reduces the amount of WSC used, shortens the reaction time, and lowers production costs, making it valuable for industrial application.

[0023] 6. Combines antibacterial and healing-promoting functions: The hemostatic sponge formula contains carboxymethyl chitosan, which itself has certain antibacterial activity. Simultaneously, collagen, as a major component of the extracellular matrix, provides a scaffold for the migration and proliferation of fibroblasts and endothelial cells, helping to promote wound healing and epithelial repair. The sponge's porous network structure can also serve as a carrier for antibacterial drugs or growth factors, achieving drug encapsulation and sustained release during use, resulting in long-lasting antibacterial, anti-inflammatory, and healing-promoting effects, reducing the risk of wound infection, and overcoming the shortcomings of existing expanded sponges that require additional antibiotic application. Attached Figure Description

[0024] Figure 1 Comparison of scanning electron microscopy (SEM) images of sponges prepared with different crosslinking agent concentrations.

[0025] Figure 2 A comparison chart of water absorption ratios of sponges prepared with different crosslinking agent concentrations.

[0026] Figure 3 Comparison of SEM images prepared for different reaction times.

[0027] Figure 4 Comparison of SEM images prepared at different reaction pH values.

[0028] Figure 5 Comparison of SEM images prepared at different reaction temperatures.

[0029] Figure 6 SEM comparison images of samples prepared with different concentrations of gelatin.

[0030] Figure 7 Comparative photographs of the macroscopic morphology of sponges with different cross-linking agent concentrations after 4 days of degradation in bacterial culture medium.

[0031] Figure 8 Photographs showing the macroscopic morphological changes of a sponge with a 10% concentration of cross-linking agent in bacterial culture medium at different degradation times.

[0032] Figure 9 SEM comparison images of sponge with 20% crosslinking agent before and after degradation.

[0033] Figure 10 SEM images comparing sponges prepared at different reaction temperatures before and after degradation.

[0034] Figure 11 This is a comparison chart of the water absorption performance of sponges in salt water of different concentrations.

[0035] Figure 12 This is a comparison chart of the water absorption performance of sponges in solutions with different pH values.

[0036] Figure 13 This is a comparison chart of the water absorption performance of sponges under different temperature conditions.

[0037] Figure 14 A grouped bar chart comparing the effects of different protease treatments on the water absorption ratio of sponges. Detailed Implementation

[0038] The present invention will be further described in detail below through specific embodiments, but the present invention is not limited to the following embodiments. Any equivalent substitutions or modifications made to the present invention by those skilled in the art without departing from the concept of the present invention should be included within the protection scope of the present invention.

[0039] I. Raw Material Sources and Performance Indicators The main raw material sources and control standards used in this invention are as follows: 1. γ-Polyglutamic acid (γ-PGA): Supplied by Nanjing Saites Biotechnology Co., Ltd., with a molecular weight ≥1000kDa, content ≥92%, appearance as a white amorphous free-flowing powder, pH (1% solution) 5.0-7.0, transmittance (5mg / mL, 400nm) ≥95.0%, and loss on drying ≤8.0%. γ-PGA is an extracellular adhesive substance synthesized through microbial fermentation. It is a polyamide formed by peptide bonds between L-glutamic acid and / or D-glutamic acid monomers through α-amino and γ-carboxyl groups. The structural formula of its repeating unit is as follows:

[0040] Where n represents the degree of polymerization, and the molecular weight is ≥1000kDa. The γ-PGA backbone contains a large number of free carboxyl groups, giving it excellent hydrophilicity and crosslinking properties.

[0041] 2. Gelatin: Supplied by Beijing Tongguang Fine Chemical Co., Ltd., it is a medical-grade raw material.

[0042] 3. Carboxymethyl chitosan: supplied by Zhejiang Aoxing Biotechnology Co., Ltd., is a medical-grade product that has undergone biosafety verification.

[0043] 4. Collagen: Provided by Beijing Luyuan Bode Biotechnology Co., Ltd., it is bovine Achilles tendon type I collagen with a content of ≥99.0% and a moisture content of ≤1.0%.

[0044] 5. Glycerin: Medical grade, provided by Zhejiang Aoxing Biotechnology Co., Ltd.

[0045] 6. Main crosslinking agent – ​​Water-soluble carbodiimide (WSC): Provided by Beijing Luyuan Bode Biotechnology Co., Ltd., with a content ≥99.0%, moisture ≤1.0%, and appearance as a white crystalline powder. Under acidic conditions, the —N=C=N— groups of WSC are activated and react with the carboxyl groups on the γ-PGA molecular chain to form a crosslinked structure. Specifically, the activated WSC combines with the carboxyl groups of the γ-PGA side chain to form an active intermediate. This intermediate further reacts with the amino or hydroxyl groups on adjacent molecular chains to form stable amide or ester crosslinking bridges, thereby transforming γ-PGA from a linear macromolecule into a three-dimensional network gel structure.

[0046] 7. First auxiliary crosslinking agent – ​​ethylene glycol diglycidyl ether: analytical grade, preferably product of Tokyo Chemical Industry Co., Ltd. (TCI), Japan.

[0047] 8. Second auxiliary crosslinking agent - ammonium chloride: analytical grade, commercially available.

[0048] II. Preparation process of hemostatic sponge Example 1. Preparation process of the optimal formulation for industrialization Measure 5000 mL of distilled water, and add 4900 mL of it to the reaction vessel. Heat and stabilize at 37°C. Add 249.60 g of polyglutamic acid and 5.72 g of collagen to the reaction vessel, set the stirring speed to 260 rpm, and stir for 20 minutes to ensure that the polyglutamic acid and collagen are completely dissolved. Then add 51.2 g of gelatin and 15.6 g of carboxymethyl chitosan to the reaction vessel, along with 39.9 g of glycerol. Continue stirring at 260 rpm for 10 minutes to ensure that all polymer components are completely dissolved.

[0049] Add 1.60 g of the first auxiliary crosslinking agent, ethylene glycol diglycidyl ether, and 1.60 g of the second auxiliary crosslinking agent, ammonium chloride, to the reaction system and allow the crosslinking reaction to proceed for 5 minutes. Then add 6.38 g of the main crosslinking agent, WSC, which has been dissolved in 100 mL of distilled water beforehand. The total mass of the crosslinking agent accounts for 2.57% of the total mass of the sponge. Observe the crosslinking reaction process. When the viscosity of the system increases significantly and stirring becomes difficult, stop stirring, keep the system at the desired temperature and allow it to stand for 40 minutes to complete the gelation process.

[0050] The obtained gel was transferred to a flat-bottomed, thin-walled petri dish and pre-frozen in a refrigerator. After the sample was completely frozen, it was dried under vacuum conditions using a freeze dryer for approximately 48 hours to obtain the absorbable polyglutamic acid composite hemostatic sponge. The resulting sponge product has a white, uniform, and fluffy appearance, and exhibits good flexibility and elasticity.

[0051] The sponge prepared in this embodiment was tested and found to have a water absorption rate of 43.6 g / g in 5 minutes, 45.1 g / g in 30 minutes, and 46.6 g / g in 1 hour.

[0052] Example 2. Effect of different crosslinking agent ratios on sponge properties Under the following conditions: a fixed polyglutamic acid concentration of 4.0%, glycerol dosage of 13% of the total molecular weight, a gelatin to polyglutamic acid mass ratio of 0.2, a carboxymethyl chitosan to polyglutamic acid mass ratio of 0.06, a reaction temperature of 37℃, and a reaction time of 40 minutes, the proportions of the three crosslinking agents were varied. The results are shown in Table 1 below.

[0053] Table 1. Water absorption ratio of sponges with different crosslinking agent ratios (g / g) mWSC:m(PGA+GLA) crosslinking agent 2: m(PGA+GLA) crosslinking agent 3: m(PGA+GLA) Q5min Q30min Q1h 0.016 0.006 0.002 39.7 43.7 45.6 0.016 0.004 0.004 40.3 46.7 55.6 0.016 0.002 0.006 37.2 38.9 43.8 0.022 0.008 0.003 33.1 34.5 37.1 0.022 0.0055 0.0055 36.5 41.3 46.7 0.022 0.003 0.008 31.2 37.6 38.9 Experiments show that the sponge produced has the best overall expansion performance when the total mass of the first auxiliary crosslinking agent and the second auxiliary crosslinking agent accounts for 50% of the mass of the main crosslinking agent WSC, and the mass ratio of the two is 1:1.

[0054] To investigate the effect of crosslinking agent concentration on the microstructure of sponges, scanning electron microscopy (SEM) was used to observe sponges prepared with different WSC concentrations (0%, 5%, 10%, 20%, 40%, relative to the total mass of γ-PGA and gelatin). Figure 1 As shown, with the increase of crosslinking agent dosage, the microporous structure of the sponge exhibits a trend of changing from loose to uniform to dense. When the WSC concentration is 10%, the sponge displays the most uniform porous network structure; when the concentration reaches 40%, the crosslinking points become too dense, the network space shrinks, and the pore structure becomes dense. The above structural changes are closely related to its water absorption performance.

[0055] like Figure 2 As shown, the water absorption ratio test of sponges with different crosslinking agent concentrations indicates that the 10% concentration sponge achieves a water absorption ratio of 118 g / g within 30 minutes, significantly higher than that of sponges without crosslinking agent (30 g / g) and those with a 40% concentration (18 g / g). Further water absorption kinetic simulations show that the water absorption rate of the 10% concentration sponge (36.1 g / min) is also optimal.

[0056] Example 3. Effect of different reaction times on sponge properties With a fixed raw material concentration of 2%, a WSC crosslinking agent concentration of 10%, a reaction temperature of 50℃, and a reaction system pH of 5, the reaction times were varied to 6 h, 12 h, 16 h, 20 h, and 30 h. SEM images of the prepared sponges are shown below. Figure 3 As shown, with the extension of reaction time, the sponge pore structure gradually becomes more uniform and complete, with the pore structure being most ideal at 20 hours. However, when the reaction time is further extended to 30 hours, the γ-PGA molecular chains are partially degraded due to prolonged high temperature, resulting in uneven pore structure and collapse. Therefore, in industrial formulations, by improving reaction efficiency and optimizing the crosslinking system, the reaction time is shortened to 40 minutes, ensuring the crosslinking effect while avoiding energy waste.

[0057] Example 4. Effect of different reaction pH on sponge properties With a fixed raw material concentration of 2%, a crosslinking agent WSC concentration of 10%, a reaction temperature of 50℃, and a reaction time of 6 hours, the pH value of the reaction system was changed to 3, 4, 5, 6, and without hydrochloric acid adjustment. Figure 4 As shown, the sponge exhibits the most uniform porous structure at pH=5, with a porosity of 47.38%. At pH=3, the strongly acidic environment leads to the degradation of γ-PGA molecular chains, significantly damaging the sponge's pore structure. Without hydrochloric acid, the WSC crosslinking activity is insufficient, and the pore structure is also less uniform. The industrial-scale formulation of this invention adopts a method that does not require additional pH adjustment. In a neutral to slightly acidic environment, it achieves efficient crosslinking with the synergistic effect of an auxiliary crosslinking agent, thus avoiding acid degradation while ensuring the crosslinking effect.

[0058] Example 5. Effect of different reaction temperatures on sponge properties With a fixed raw material concentration of 2%, a WSC crosslinking agent concentration of 10%, a reaction system pH of 5, and a reaction time of 6 hours, the reaction temperatures were varied at 40℃, 50℃, 60℃, 70℃, and 80℃. SEM images (…) Figure 5 The results showed that the sponge's pore structure was most uniform and intact at 50℃; when the temperature rose to 80℃, the pore structure was completely destroyed, exhibiting an irregular lamellar structure. Water absorption rate tests showed that the sponge prepared at 50℃ had the highest water absorption rate after 30 minutes (53.4 g / g), while the sponge prepared at 80℃ showed a decrease to 12.6 g / g. Considering industrialization efficiency and structural stability, 37℃ was selected as the preferred cross-linking reaction temperature.

[0059] Example 6. Effect of different gelatin addition amounts on sponge properties Based on the above optimized conditions, the concentration of polyglutamic acid in the solution was fixed at 4.8%, the ratio of WSC crosslinking agent to polymer mass was 0.02, the auxiliary crosslinking agents each accounted for 25% of the mass of the main crosslinking agent, the amount of glycerol was 13% of the total polymer mass, the mass ratio of carboxymethyl chitosan to polyglutamic acid was 0.06, the reaction temperature was 37℃, and the reaction time was 40 minutes. The mass ratio of gelatin to polyglutamic acid (mGLA:mPGA) was changed to 0.18, 0.20, 0.22, 0.24, and 0.26. The results are shown in Table 2 below.

[0060] Table 2. Water absorption ratio of sponges with different amounts of gelatin added (g / g) mGLA:mPGA 0.18 0.20 0.22 0.24 0.26 Q5min 47.1 61.2 58.4 51.2 48.3 Q30min 53.6 81.7 77.9 72.6 66.3 Q1h 57.5 88.9 86.3 81.7 70.5 The results showed that the sponge expansion ratio was highest when mGLA:mPGA = 0.20. (SEM image) Figure 6 The results show that as the gelatin content increases, the loose network structure of the sponge gradually becomes denser. This is because there are strong hydrogen bonds between gelatin molecules; adding an appropriate amount helps stabilize the cross-linked network, but adding too much will make the network too dense, shorten the chain segments, and reduce the water absorption capacity. Therefore, a mGLA:mPGA ratio of 0.20 is preferred.

[0061] Example 7. Effect of different polyglutamic acid concentrations on sponge properties The ratio of WSC (the main crosslinking agent) to polymer mass was fixed at 0.02, each auxiliary crosslinking agent accounted for 25% of the main crosslinking agent's mass, glycerol was 13% of the polymer mass, the ratio of gelatin to polyglutamic acid was 0.20, the ratio of carboxymethyl chitosan to polyglutamic acid was 0.06, the reaction temperature was 37℃, and the reaction time was 40 minutes. The mass concentration of polyglutamic acid in the solution was varied, and the results are shown in Table 3 below.

[0062] Table 3. Water absorption ratio (g / g) of sponges at different polyglutamic acid concentrations. PGA concentration in solution (%) 3.2 4.0 4.8 5.6 6.4 Q5min 21.7 48.7 61.2 68.9 53.2 Q30min 28.9 51.6 81.7 99.7 60.1 Q1h 25.3 60.1 88.9 105.6 63.9 Considering both expansion performance and gel molding processability, a polyglutamic acid concentration of 4.8% in the solution is optimal. If the concentration is too low, cross-linking is insufficient, and the sponge easily dissolves after absorbing water; if the concentration is too high, the solution viscosity is too high, making molding difficult and the network too dense, thus reducing expansion performance.

[0063] Example 8. Water absorption performance test of sponge in different media To evaluate the performance of the sponge in simulated physiological environments, the water absorption ratio of the sponge prepared in Example 1 was tested in different media.

[0064] 1. Water absorption properties of salt solutions of different concentrations: such as Figure 11 As shown, the water absorption rate of the sponge decreased significantly as the salt solution concentration increased from 0% to 0.9%. In a 0.9% physiological saline environment, the water absorption rate was approximately 8.4 g / g after 30 minutes. This is because salt ions shield the repulsive effect of like charges in the polymer network, thus limiting network expansion.

[0065] 2. Water absorption properties in solutions with different pH values: such as Figure 12 As shown, the sponge has a high and stable water absorption ratio within the pH range of 5-7; in an alkaline solution with pH=9.81, the water absorption ratio still maintains a stable increase, indicating that the sponge has good acid and alkali adaptability.

[0066] 3. Water absorption performance at different temperatures: such as Figure 13 As shown, within the temperature range of 40℃-80℃, the sponge has the highest water absorption rate at 50℃, reaching 32.3g / g after 250 minutes. Further increases in temperature lead to decreased network elasticity or thermal degradation, resulting in a decrease in water absorption rate.

[0067] Example 9. Degradation performance test of sponge 1. Enzyme degradation test The sponge prepared in Example 1 was placed in trypsin or papain solution (enzyme activity 2450 U / mL) to test the effect of different enzyme treatment conditions on the sponge's water absorption performance. Figure 14 As shown, papain exhibits the most significant degradation effect on sponges at pH 4-5; sponges prepared at pH 4 show a sharp decrease in water absorption capacity after papain treatment. Trypsin, on the other hand, shows the strongest activity at pH 3-4. This indicates that the amide bonds in sponges can be effectively broken by proteases, achieving enzymatic degradation.

[0068] 2. Bacterial culture medium degradation test Prepare a bacterial culture medium containing microorganisms such as Bacillus subtilis, and immerse sponges prepared with different concentrations of cross-linking agents (0%, 5%, 10%, 20%, 40%) into it. Figure 7The macroscopic morphology of sponges with different cross-linking agent concentrations was shown after 4 days of degradation. The sponge without cross-linking agent was almost completely dissolved in the solution, while the sponge with 40% cross-linking agent still maintained a complete gel-like morphology, indicating that the higher the cross-linking density, the stronger the degradation resistance.

[0069] Figure 8 The continuous morphological changes of a 10% concentration crosslinking agent sponge at 4, 7, 8, and 10 days of degradation were shown, clearly presenting the entire process from complete gel → surface dispersion → complete dissolution.

[0070] Figure 9 These are SEM images comparing the sponge with 20% crosslinking agent before and after degradation. Before degradation, the sponge exhibits a regular porous network structure. After 10 days of degradation, the pore structure is significantly disrupted, the pore walls thicken, and some areas collapse and fuse.

[0071] Figure 10 SEM images comparing sponges prepared at different reaction temperatures (40℃, 50℃, 60℃, 70℃) before and after degradation. The sponge prepared at 50℃ has the most uniform and dense structure, and the degree of structural damage after degradation is relatively minor; while the sponges prepared at higher temperatures have poorer structural stability, and the changes in pore structure after degradation are more significant.

[0072] Table 4 summarizes the mass degradation rate of sponges with different cross-linking agent concentrations at different time points in bacterial culture medium.

[0073] Table 4. Degradation rate (%) of sponges with different cross-linking agent concentrations in bacterial culture medium Degradation time (days) No cross-linking agent added 5% WSC 10% WSC 20% WSC 40% WSC 4 85 65 48 35 23 6 92 70 55 43 28 8 — 73 62 50 35 10 — — 72 59 45 12 — — — 64 50 Experimental results showed that all samples achieved a mass loss of over 50% within 12 days, demonstrating good biodegradability. By adjusting the amount of cross-linking agent and gelatin content, the degradation rate of the sponge can be effectively controlled to meet the application needs of different clinical scenarios.

[0074] III. Performance Characterization Methods 1. Water absorption ratio determination: The nylon bag method was used. A certain mass of ( The dry sponge is placed in a nylon bag, immersed in deionized water or physiological saline, removed at regular intervals, drained of surface moisture, and then weighed. ), water absorption ratio .

[0075] 2. Water absorption kinetics simulation: The Voigt model was used to fit the sponge swelling process, and the formula is as follows: ,in To balance the water absorption ratio, This is the relaxation time.

[0076] 3. Pore structure characterization: The cross-sectional morphology of the sponge was observed using a scanning electron microscope (SEM), and parameters such as porosity, pore diameter (D), diameter of an equal-area circle (Dea), diameter of an equal-circumference circle (Dc), and pore wall thickness (LD) were calculated using ImageJ software and stereoscopic principles.

[0077] 4. Degradation rate calculation: Place the sponge sample in a degradation medium (enzyme solution or bacterial culture medium), periodically remove it, freeze-dry it, and weigh it. ), degradation rate ,in This represents the initial mass.

[0078] IV. Conclusion Based on the above embodiments and test results, this invention provides an absorbable composite hemostatic sponge prepared using polyglutamic acid as the core raw material, combined with gelatin and carboxymethyl chitosan, and through a WSC / ethylene glycol diglycidyl ether / ammonium chloride ternary crosslinking system. This sponge has the following outstanding advantages: 1. Excellent water absorption and swelling properties; the water absorption rate of deionized water can reach more than 80g / g within 30 minutes, meeting the clinical hemostasis requirements for rapid fluid absorption, swelling and compression. 2. Excellent tissue adhesion: The addition of collagen allows the sponge to adhere to the bleeding wound, working synergistically with expansion and compression to achieve a dual hemostatic effect; 3. Suitable biodegradation rate: γ-polyglutamic acid and collagen can be degraded into non-toxic small molecules such as amino acids in vivo, with good biocompatibility and no side effects from degradation products; 4. Uniform and controllable microporous structure; porosity, pore size and mechanical flexibility can be controlled by adjusting the preparation process parameters to adapt to different wound packing needs; 5. The preparation process is simple and cost-controllable. The reaction conditions are mild (37℃, 40 minutes), requiring no strong acids or bases or high temperature and pressure, making it easy to achieve industrial-scale production.

[0079] In summary, the absorbable polyglutamic acid composite hemostatic sponge of the present invention has broad application prospects in the field of packing hemostasis, such as clinical surgery and trauma emergency care.

Claims

1. An absorbable arterial hemostatic sponge, characterized in that, It is prepared from raw materials containing the following components through a cross-linking reaction and freeze-drying: γ-polyglutamic acid; gelatin; Carboxymethyl chitosan; Collagen; glycerin; and crosslinking agent system; The crosslinking agent system includes a main crosslinking agent, a first auxiliary crosslinking agent, and a second auxiliary crosslinking agent. The main crosslinking agent is a water-soluble carbodiimide, the first auxiliary crosslinking agent is a glycidyl ether crosslinking agent that can crosslink with gelatin, and the second auxiliary crosslinking agent is an auxiliary agent that can promote the crosslinking of water-soluble carbodiimide with γ-polyglutamic acid.

2. The absorbable arterial hemostatic sponge according to claim 1, characterized in that, The first auxiliary crosslinking agent is ethylene glycol diglycidyl ether, and the second auxiliary crosslinking agent is ammonium chloride.

3. The absorbable arterial hemostatic sponge according to claim 2, characterized in that, The proportions of the raw materials must meet at least one of the following requirements: The mass ratio of the main crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.02:1; The mass ratio of the first auxiliary crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.01 : 1; The mass ratio of the second auxiliary crosslinking agent to the sum of the masses of γ-polyglutamic acid and gelatin is 0.005 : 1; The mass concentration of γ-polyglutamic acid in the reaction solution was 4.8%-5.1%; The mass ratio of glycerol to the sum of the masses of γ-polyglutamic acid and gelatin is 0.13 : 1; The mass ratio of gelatin to γ-polyglutamic acid was 0.2:1; The mass ratio of carboxymethyl chitosan to γ-polyglutamic acid is 0.06:1; The mass ratio of collagen to γ-polyglutamic acid is 0.01:1-0.05:

1.

4. The absorbable arterial hemostatic sponge according to claim 2, characterized in that, The total mass of the first auxiliary crosslinking agent and the second auxiliary crosslinking agent accounts for 40%-60% of the main crosslinking agent, and the mass ratio of the first auxiliary crosslinking agent to the second auxiliary crosslinking agent is 0.8:1-1.2:

1.

5. The absorbable arterial hemostatic sponge according to claim 1, characterized in that, The molecular weight of γ-polyglutamic acid is ≥1000kDa.

6. The absorbable arterial hemostatic sponge according to claim 1, characterized in that, The hemostatic sponge has a uniform microporous structure with a porosity of 5%-48% and a pore size range of 4μm-170μm.

7. The absorbable arterial hemostatic sponge according to claim 1, characterized in that, The hemostatic sponge is degradable in protease-containing buffer or bacterial culture medium, with a degradation rate of ≥55% after 6 days and ≥50% after 12 days in bacterial culture medium.

8. A method for preparing an absorbable arterial hemostatic sponge as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Step S1: Dissolve γ-polyglutamic acid, gelatin, collagen, carboxymethyl chitosan and glycerol in water and stir until homogeneous to obtain a polymer solution; Step S2: Add the first auxiliary crosslinking agent and the second auxiliary crosslinking agent to the obtained polymer solution to carry out the first crosslinking reaction; Step S3: Then add the main crosslinking agent to carry out the second crosslinking reaction and form a gel; Step S4: Pre-freeze the obtained gel to form a solid shape, and then use freeze-drying method to vacuum dry it to obtain a hemostatic sponge.

9. The preparation method according to claim 8, characterized in that, The water temperature in step S1 is 35℃-40℃; the total cross-linking reaction time in steps S2 and S3 is 30-60 minutes.

10. An absorbable arterial hemostatic sponge as described in any one of claims 1 to 7, characterized in that, Application in the preparation of medical dressings or hemostatic materials for hemostasis during clinical surgery and trauma emergency treatment.

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