Highly effective hemostatic materials for inhibiting overt and covert bleeding, their preparation and application

Abstract

This invention provides a highly efficient hemostatic material for inhibiting both overt and covert bleeding, and its preparation and application. The preparation of this hemostatic material includes the following steps: (1) inoculating bacterial colonies into a culture medium for fermentation to obtain a hydrogel-like bacterial cellulose membrane; shearing, homogenizing, and freeze-drying the bacterial cellulose membrane to obtain bacterial cellulose nanofibers; (2) dispersing the bacterial cellulose nanofibers and then oxidizing them to obtain oxidized bacterial cellulose nanofibers; (3) mixing the oxidized bacterial cellulose nanofibers with tranexamic acid at a weight ratio of 3:100, and preparing an oxidized bacterial cellulose nanofiber composite loaded with tranexamic acid using an EDC / NHS reaction, which is the hemostatic material. The hemostatic material of this invention can achieve the dual effect of inhibiting both overt and covert bleeding in vivo, and while inhibiting covert bleeding, it also significantly improves the effect of inhibiting overt bleeding.

Description

Technical Field

[0001] This invention belongs to the field of medical hemostatic materials technology, specifically relating to a highly effective hemostatic material for inhibiting overt and covert bleeding, and its preparation and application. Background Technology

[0002] In military, sports, and medical fields, injuries such as trauma, soft tissue lacerations, penetrating wounds, and burns occur frequently. These wounds often lead to external or internal bleeding, causing pain and increasing the risk of infection, which in severe cases can even threaten the lives of the wounded. Therefore, the development of hemostatic materials that are highly effective in stopping bleeding, providing long-lasting pain relief, preventing and eliminating wound infection, and effectively promoting wound healing is particularly urgent. Rapid and effective hemostasis is also a key factor in improving the survival rate of wounded soldiers in combat and trauma.

[0003] Studies have shown that blood coagulation is a process in which a series of coagulation factors are successively enzymatically activated, specifically including the formation of prothrombin activator, thrombin formation, and fibrin formation. There are three main types of coagulation mechanisms: (1) direct activation or participation in the coagulation system; (2) enrichment of coagulation components at the wound site through physical and chemical pathways such as water absorption, thereby stimulating physiological hemostasis; and (3) physical closure of blood vessels through strong adhesion. Based on these coagulation mechanisms, researchers have developed a series of medical materials for hemostasis.

[0004] Commonly used hemostatic materials can be categorized as follows: 1) Materials with strong hydrophilic and absorbent properties, such as cotton fabrics like gauze, inorganic porous materials like porous zeolite, and starch; 2) Hemostatic materials utilizing special chemical actions, such as electrostatic attraction. Since blood cells carry a negative charge, positively charged polysaccharides can be introduced onto the fabric surface, promoting blood coagulation through electrostatic interaction; 3) Polypeptide-based hemostatic materials, such as fibrin and collagen. Collagen can activate the activity of some blood clotting factors, guide platelet attachment, and produce release reactions and aggregation. It can also fill the gaps caused by mechanical compression of damaged blood vessels; 4) Composite materials, such as polysaccharide-peptide composites, and composite materials of hemostatic dressings and hemostatic gauze or bandages.

[0005] While ordinary cotton fabrics such as gauze and hemostatic bandages have some hemostatic effect, cotton fabrics have a very high blood absorption rate. When bandaging bleeding wounds, the high blood absorption of gauze can cause patients to lose even more blood. Excessive blood loss can lead to fainting, shock, or even death. Therefore, researching and developing hemostatic materials with short hemostatic time and low blood absorption during the hemostatic process is of great significance.

[0006] Currently, gelatin sponges are commonly used hemostatic materials in clinical practice. However, these medical dressings generate a large amount of medical waste and have poor absorbency. Furthermore, removing these dressings can cause secondary injury to patients. Therefore, it is particularly important to develop hemostatic materials that have hemostatic effects, low blood loss, and rapid hemostasis.

[0007] As is well known, bleeding is divided into overt and covert bleeding. However, current hemostatic materials mainly focus on suppressing visible overt bleeding, with very little attention paid to covert bleeding. Covert bleeding is internal bleeding that is difficult to avoid after trauma or surgery, directly leading to a sharp drop in hemoglobin, which in turn causes anemia and even death.

[0008] Research Report [1] In elderly patients undergoing surgery for intertrochanteric fractures of the femur, those treated with proximal femoral antirotation intramedullary nailing experienced an average intraoperative blood loss of (48.9±2.8) mL, an average postoperative overt blood loss of (62.3±3.8) mL, and an average occult blood loss of (385.0±6.2) mL. Clearly, occult blood loss was significantly higher than overt blood loss. (Yoji Ogur et al.) [2] This report describes 107 patients who underwent anterior cervical fusion for degenerative spine disease, with occult blood loss totaling 261 mL, accounting for 50% of the total blood loss. Therefore, how to better suppress occult bleeding while inhibiting overt bleeding is a crucial issue that hemostatic materials must address.

[0009] The gold standard for improving occult bleeding in clinical practice is whole blood or blood component transfusion, as well as the use of fibrinogen concentrate or recombinant clotting factors. However, these methods carry risks such as immunogenicity and contamination. Therefore, occult blood loss is a significant risk factor that has a substantial impact on patients' postoperative quality of life. Reducing postoperative occult blood loss can effectively promote postoperative functional recovery and improve patients' quality of life.

[0010] Currently, the most common clinical approach to controlling occult bleeding is intravenous infusion or local immersion in tranexamic acid (TXA), widely used in orthopedic surgery, brain surgery, and postpartum hemorrhage. Multiple clinical trials in various settings have demonstrated that tranexamic acid can reduce occult blood loss, transfusion rates, and bleeding-related mortality. However, when using tranexamic acid to suppress occult bleeding, the effectiveness of hemostatic materials in suppressing overt bleeding decreases. Improving the effectiveness of tranexamic acid hemostatic materials in suppressing overt bleeding, and developing a medical hemostatic material that effectively controls occult bleeding while also providing good overt hemostasis and rapid hemostasis, has become a major challenge in hemostatic material development.

[0011] Patent document CN 103394078 A discloses a biodegradable hemostatic agent for emergency treatment of trauma, which is characterized by autonomous pressure and multi-target synergistic hemostasis. The agent comprises 10-90% biodegradable material, 1-40% thrombin, 1-40% fibrinogen, 1-40% coagulation factor, 1%-20% tranexamic acid, 1%-20% desmopressin, 0.5%-20% calcium salt, 0.1-5% local anesthetic analgesic, and 0.01-2% nano-silver. The biodegradable material is gelatin, microfibrillary collagen, chitosan, collagen, or biodegradable cellulose, which can inhibit various types of bleeding in a short time. However, the biodegradable hemostatic agent in this patent has the problems of high bleeding volume and insufficient inhibition of overt hemostasis. In a rat model of bleeding after middle lobe resection of the liver, the hemostatic effect of this biodegradable hemostatic agent showed that when the bleeding time was within 200 seconds, the bleeding volume reached more than 1.01g. In rats with coagulation disorders, the hemostatic effect on femoral artery incisions showed that when the bleeding time was around 180 seconds, the bleeding volume reached 1.76g. Therefore, while this tranexamic acid hemostatic material achieves latent hemostasis, it suffers from poor inhibition of overt hemostasis and high overt bleeding volume.

[0012] Patent document CN 113667706 A discloses a tranexamic acid-crosslinked porous starch hemostatic material, prepared by enzymatic hydrolysis of starch to create pores, tranexamic acid protected by BOC undergoing an acyl chloride reaction, and esterification of starch with BOC-carbamoyl chloride. The raw materials include starch, BOC-tranexamic acid, thionyl chloride, and amylase. In a mouse tail amputation model, the hemostatic effect of this material showed that the free bleeding volume within 10 seconds reached 0.7g, indicating a high overt bleeding volume. Therefore, while this tranexamic acid-crosslinked porous starch hemostatic material achieves latent hemostasis, it still suffers from a high overt bleeding volume.

[0013] It is evident that current methods using tranexamic acid for latent hemostasis generally suffer from poor efficacy in suppressing overt bleeding. These tranexamic acid hemostatic materials result in higher overt bleeding volumes and slower hemostasis. Therefore, developing a tranexamic acid hemostatic material that combines both latent and overt hemostasis to address the shortcomings of tranexamic acid in overt hemostasis, reduce overt bleeding, and achieve faster hemostasis has become an urgent technical challenge.

[0014] The cited references are as follows:

[0015] [1] Zhang, PX, et al., Clinical analysis of obvious and hidden bloodloss in inter-trochanter fracture patients treated with proximal femoral nail anti-rotation and dynamic hip screw. Beijing Da Xue Xue Bao Yi Xue Ban, 2012.44(6):p.891-894.

[0016] [2]Ogura,Y.,et al.,Hidden blood loss following 2-to 3-level posteriorlumbar fusion. Spine J, 2019.19(12):p.2003-2006. Summary of the Invention

[0017] The present invention aims to solve the aforementioned technical problems, thereby providing a highly effective hemostatic material for inhibiting both overt and covert bleeding, as well as its preparation and application. The technical objective of this invention is twofold: firstly, to address the issue that existing tranexamic acid-functionalized hemostatic materials primarily target covert bleeding, significantly reducing their effectiveness in controlling overt bleeding; and secondly, to address the problems of high overt bleeding volume and insufficient overt hemostatic effect associated with existing tranexamic acid hemostatic materials.

[0018] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0019] This invention first provides a method for preparing a hemostatic material that effectively inhibits overt and covert bleeding, comprising the following steps:

[0020] (1) Colonies were inoculated into a culture medium for fermentation culture to obtain a hydrogel-like bacterial cellulose membrane on the gas-liquid surface; the bacterial cellulose membrane was sheared, homogenized and freeze-dried to obtain bacterial cellulose nanofibers;

[0021] (2) Bacterial cellulose nanofibers were dispersed and then oxidized to obtain oxidized bacterial cellulose nanofibers.

[0022] (3) Oxidizing bacterial cellulose nanofibers and tranexamic acid are mixed at a weight ratio of 3:100, and tranexamic acid-loaded oxidizing bacterial cellulose nanofibers composite is prepared by EDC / NHS reaction, which is the hemostatic material.

[0023] The method provided by this invention first synthesizes bacterial cellulose membranes and prepares them into short nanofibers. Then, it obtains degradable oxidized bacterial cellulose nanofibers through TEMPO chemical oxidation. The nitric oxide free radicals in the ammonium oxychloride form of the TEMPO structure selectively oxidize the primary alcohols of the BNC side chain to carboxyl groups, which then form amide bonds with the amino groups on tranexamic acid, thereby constructing a short fiber-based hemostatic material capable of simultaneously inhibiting overt and covert bleeding. Experiments have shown that the nanofibers provided by this invention can be prepared into various forms such as suspensions, gels, sponges, and powders to meet the hemostatic needs of different application scenarios. Simultaneously, this nanofiber-based hemostatic material exhibits excellent antibacterial effects, promotes the proliferation of rat fibroblasts, promotes the adhesion of platelets and whole blood cells in vitro, and promotes coagulation reactions. In rat tail hemorrhage models, liver hemorrhage models, and rectus abdominis defect hemorrhage models, it has a significant and excellent effect in inhibiting overt bleeding, and also has a significant and excellent effect in inhibiting covert bleeding in the rectus abdominis hemorrhage model. It is a highly efficient hemostatic material that simultaneously inhibits overt and covert bleeding.

[0024] Furthermore, the colony described in step (1) is *Acetobacter xylodis*.

[0025] Furthermore, the fermentation medium in step (1) consists of 100 g / L D-fructose, 5 g / L peptone and 3 g / L yeast extract, with a pH of 5.0.

[0026] Furthermore, the culture conditions described in step (1) are constant temperature culture at 30℃ for 7 days.

[0027] Furthermore, the homogenization operation in step (1) is to use a homogenizer to process the product at a speed of 15000 rpm for 15 minutes.

[0028] Furthermore, the preparation method of the oxidative bacterial cellulose nanofibers in step (2) is as follows: the freeze-dried BNC short fibers are redispersed in sodium phosphate buffer, tetramethylpiperidine oxide and NaClO2 are dissolved in the suspension, NaClO is added dropwise to the sodium phosphate buffer solution, and then the suspension is added immediately. The mixture is magnetically stirred at 50°C for 48 hours. After washing and freeze-drying, the oxidative bacterial cellulose nanofibers are obtained.

[0029] Furthermore, the EDC / NHS reaction in step (3) includes the following steps: dispersing OBNC short fibers in MES buffer, adding NaCl, NHS and EDC in sequence, then adding tranexamic acid, and stirring at 30°C for 12 hours to obtain the product.

[0030] A second objective of this invention is to provide a hemostatic material that can effectively inhibit overt and covert bleeding, prepared by the method described in any of the preceding claims.

[0031] Furthermore, the hemostatic material includes emulsion, gel, sponge, or powder forms.

[0032] A third objective of this invention is to provide the application of a highly efficient hemostatic material prepared by any of the methods described above in simultaneously inhibiting overt and covert bleeding.

[0033] The beneficial effects of this invention are as follows:

[0034] (1) This invention provides a biodegradable tranexamic acid-functionalized bacterial cellulose short fiber. The short fiber has micron and nano-sized pores, a water absorption rate of over 96%, can rapidly activate the coagulation reaction to inhibit overt bleeding, and can continuously release tranexamic acid and diffuse into the tissue to reduce the activity of plasmin and inhibit occult bleeding.

[0035] (2) The short fiber hemostatic material provided by the present invention has excellent antibacterial effect. In vitro, its antibacterial rate against Escherichia coli and Staphylococcus aureus is about 2.36 times and 1.57 times that of chitosan, respectively.

[0036] (3) The short fiber hemostatic material provided by this invention has obvious dual effects of inhibiting both overt and covert bleeding. In in vitro blood experiments, the short fiber hemostatic material has significantly excellent procoagulant properties. In coagulation experiments of rat tail amputation and liver injury bleeding models, the short fiber hemostatic material showed low bleeding volume, and at the same time inhibited covert bleeding in the rectus abdominis muscle defect bleeding model, with hemoglobin levels rapidly increasing from 128±5.5g / L to 165±2.6g / L within 4 days. Attached Figure Description

[0037] Figure 1 The diagram below illustrates the experimental process; (a) a schematic diagram of the bacterial BNC production process; (b) a schematic diagram of the material preparation process; (c) three different complexes i, ii, and iii that may be constructed by different proportions of OBNC and TXA, where i represents the complex that may be formed when the proportion of carboxyl groups on OBNC is greater than the proportion of amino groups on TXA, ii represents the complex that can be grafted with the amino groups on TXA in a one-to-one ratio, and iii represents the complex that can be further combined with its own carboxyl groups when the proportion of amino groups on TXA is much greater than the proportion of carboxyl groups on OBNC. Figure 2The physicochemical properties of the materials obtained in the examples are characterized as follows: (a) Four different morphologies of OBNC nanofibers: (i) representing a uniform suspension, (ii) representing a gel, (iii) representing a sponge, and (iv) representing powder; (b) Infrared spectral analysis of OBNC, OBNC-TXA1, OBNC-TXA2, OBNC-TXA3, and TXA; (c) XPS analysis of OBNC, OBNC-TXA1, OBNC-TXA2, OBNC-TXA3, and TXA; (d) SEM images of OBNC, OBNC-TXA1, OBNC-TXA2, OBNC-TXA3, and TXA for observing their microstructure.

[0038] Figure 3 This study analyzed the in vitro blood compatibility and procoagulant properties. A, B, C, and D represent short fibers in four different sponge states: OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3, respectively. (a) shows a macroscopic image of the red blood cell solution in contact with the material, indicating immediate absorption by the sponge. (b) shows a macroscopic image of the red blood cell solution after 1 hour of contact with the material, followed by the addition of physiological saline (except for the positive control, which used ultrapure water). (c) shows a macroscopic image of the sample after incubation at 37°C for 1 hour with physiological saline / ultrapure water, followed by centrifugation. It shows that red blood cells in materials A, B, C, and D, as well as the negative control group, did not show significant rupture, while red blood cells in the positive control group ruptured. (d) Hemolysis rate; (e) Changes in absorbance during whole blood coagulation; (f) Plasma recalcification kinetics curve; (g) Plasma recalcification time, where HM... time refers to the time it takes for the absorbance value to reach half of its maximum value; # indicates no significant difference (p>0.05), * indicates a significant difference (p<0.05).

[0039] Figure 4 For in vitro adhesion performance testing of blood cells; (a) shows a schematic diagram of the material incubating with platelet-rich plasma (PRP); (b, c, d) show SEM images of the material after incubation with platelets, erythrocytes, and whole blood cells, respectively; (e, f, g) show the number of platelets, erythrocytes, and whole blood cells counted based on the SEM images, respectively; # indicates no significant difference, p>0.05, * indicates significant difference, p<0.05.

[0040] Figure 5The results are for bacterial performance and cell compatibility; where A, B, C, and D represent short fibers in four different sponge states: OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3, respectively; (a) shows a schematic diagram of the antibacterial properties of the material tested by the contact method; (b) shows a macroscopic image of the colonies after plating; (c) shows the fluorescence image of live cells stained after co-culturing the material extract with L929 cells; (d) shows the antibacterial rate obtained by statistically analyzing the area of ​​the colonies using Image-J based on the macroscopic image of the colonies; (e) shows the absorbance value of the CCK-8 experiment after co-culturing the material extract with L929 cells; # indicates no significant difference (p>0.05), * indicates a significant difference (p<0.05).

[0041] Figure 6 This study evaluates the overt and covert hemostatic effects in vivo. A, B, C, and D represent four different states of short-fiber sponges: OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3, respectively. (a, b, c) show macroscopic images of the materials after blood absorption in a rat tail hemorrhage model, a rat liver injury hemorrhage model, and a rectus abdominis muscle defect hemorrhage model, respectively. (d) shows a macroscopic image of the rat rectus abdominis muscle defect hemorrhage model one week after hemostasis with different materials. (e, f, g) represent three hemorrhage models treated with four different nanofiber sponges for hemostasis. The statistical analysis of overt blood loss; (h) shows a schematic diagram of treating the bleeding site with tranexamic acid-functionalized short fibers, which promotes coagulation and reduces occult blood loss by inhibiting plasmin activity and promoting fibrin activation and aggregation; (i) shows the change in hemoglobin on the fourth day compared to the first day after hemostasis with four different nanofiber sponges in a rat rectus abdominis hemorrhage model, which indirectly reflects the occult blood loss. The control group was the one whose gauze was removed after hemostasis; # indicates no significant difference, p>0.05, * indicates a significant difference, p<0.05. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0043] The codes and their meanings used in the following embodiments are as follows:

[0044] BNC: Bacterial cellulose; OBNC: Oxidized bacterial cellulose; TEMPO: Tetramethylpiperidine oxide; TXA: Tranexamic acid; NHS: N-hydroxythiosuccinimide; EDC: N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide.

[0045] Example 1

[0046] I. Experimental Materials and Methods

[0047] 1. Experimental materials and their sources

[0048] Unless otherwise specified, all chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). L929 fibroblasts were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). D-fructose was purchased from Adamas by Shanghai Titan Technology Co., Ltd. (Shanghai, China); fetal bovine serum (FBS), Dulbecco modified Eagle (DMEM) high-glucose medium, double antibiotics (10000 U / mL penicillin, 10000 μg / mL streptomycin), and 0.25% Trypsin-EDTA (1x) were all purchased from Gibco by Shanghai Titan Technology Co., Ltd. (Shanghai, China); Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); calcium yellow-green reagent was purchased from Shanghai Yeasen Biotechnology Co., Ltd. (Shanghai, China); clean-grade SD rats (approximately 200g) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China).

[0049] 2. Construction of bacterially synthesized cellulose and tranexamic acid-functionalized cellulose

[0050] (1) Bacterial synthesis and purification of hydrogel-like BNC membranes

[0051] Colonies of *Acetobacter xylose* were inoculated into a high-temperature, high-pressure sterilized fermentation medium (100 g / L D-fructose, 5 g / L peptone, and 3 g / L yeast extract, pH adjusted to 5.0). After inoculation, the medium was incubated at 30°C for approximately 7 days, resulting in a hydrogel-like bacterial cellulose membrane (BNC membrane) on the gas-liquid surface. The purification process involved boiling the membrane in 1% NaOH solution (80°C, w / v) for 4 hours, followed by repeated replacement with ultrapure water until the pH was neutral. The membrane was then subjected to five high-temperature, high-pressure treatments in ultrapure water to meet the implantation standards for endotoxin content in Class III medical devices.

[0052] (2) Preparation of biodegradable OBNC nanofibers and functionalization with tranexamic acid

[0053] First, the hydrogel-like BNC membrane was cut into small pieces with scissors, placed in water, and homogenized in a homogenizer (IKA T-25, Staufen, Germany) at 15,000 rpm for 15 minutes. After freeze-drying, BNC nanofibers were obtained.

[0054] Degradable OBNC nanofibers were then prepared using a TEMPO (tetramethylpiperidine oxide) selective oxidation method. The specific method is as follows: 0.1 g of lyophilized BNC nanofibers were redispersed in 30 mL of sodium phosphate buffer (0.05 M, pH 6.86). TEMPO (0.1 mmol / g dry BNC) and NaClO2 (17 mmol / g dry BNC) were dissolved in the suspension. 0.2 mL of NaClO was added dropwise to 10 mL of sodium phosphate buffer (0.05 M, pH 6.86), and then immediately added to the suspension. Finally, the suspension was magnetically stirred at 50 °C for 48 hours. The product was collected and washed three times by centrifugation with copious amounts of water. After lyophilization, degradable OBNC nanofibers were obtained.

[0055] OBNC-TXA was prepared using a modified EDC / NHS-catalyzed amide bond formation composite method. Specifically, 0.3 g of OBNC short fibers were dispersed in 120 mL of 0.05 M MES buffer (pH adjusted to 5.5), followed by the sequential addition of NaCl (0.25 M), NHS (0.03 M), and EDC (0.06 M). The mixture was then dispensed into three vials, with 10 mg, 20 mg, and 30 mg of TXA added to each vial, respectively. The mixture was stirred at 30 °C for 12 h. The carboxyl content of the prepared OBNC was determined to be 1.15 mmol / g by conductivity titration. Three different nanofiber composites were prepared by adding 0.1 g of OBNC to TXA at ​​three different concentrations (10 mg, 20 mg, and 30 mg), designated as OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3.

[0056] Four different materials (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3) were suspended at a concentration of 1% (w / v), sonicated for 5 min, and then 0.5 mL was placed in the wells of a 24-well plate and freeze-dried for subsequent physicochemical property analysis, microscopic morphology observation, antibacterial performance evaluation, in vitro coagulation performance, in vitro cytotoxicity, and in vivo hemostatic performance analysis.

[0057] 3. Characterization of the physicochemical properties of nanofibers

[0058] A 1% (w / v) suspension of OBNC nanofibers was prepared and sonicated for 5 min using a cell disruptor to obtain gel-like OBNC nanofibers. This was then diluted 100-fold with water and sonicated again to obtain a diluted suspension of OBNC nanofibers. The 1% (w / v) OBNC nanofibers were freeze-dried to obtain sponge-like nanofibers, which were then pulverized to obtain powdered nanofibers. After freeze-drying, the chemical structure and composition of the samples were analyzed by ATR-FTIR spectroscopy. XPS spectra in the range of 150–600 eV and N1s 392–410 eV were scanned and recorded using X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, Waltham, Massachusetts). After freeze-drying, the samples were mounted on a sample stage, sputter-coated with gold, and their microstructure was observed using a field emission scanning electron microscope (Sirion 200, FEI, Hillsboro, Oregon, USA).

[0059] 4. Evaluation of the in vitro coagulation properties of nanofibers

[0060] (1) Preparation of blood and materials

[0061] The evaluation of in vitro coagulation performance followed the methods reported in the literature, specifically as follows: Fresh blood was collected from the heart of SD rats and mixed with 3.2% (w / v) sodium citrate to prevent coagulation, then stored at 4°C for later use. 5 mL of whole blood was centrifuged at 100 g for 10 minutes, and the supernatant was platelet-rich plasma (PRP), used for platelet adhesion experiments. The precipitate was diluted to 5 mL with physiological saline to obtain the erythrocyte solution, used for hemolysis rate and erythrocyte adhesion analysis. Platelet-poor plasma (PPP) was obtained by centrifuging whole blood at 3000 g for 5 minutes for plasma recalcification kinetic analysis.

[0062] (2) Hemolysis rate

[0063] Add 120 μL of red blood cell solution to the surface of the material and incubate at 37°C for 1 hour. Add 2 mL of physiological saline (2 mL of sterile water for the positive control) and continue incubation for another hour. Gently shake and transfer the mixture to centrifuge tubes, centrifuge at 660 g for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 550 nm using a microplate reader.

[0064] The formula for calculating hemolysis rate is: Hemolysis rate (%) = (ODt - ODn) / (ODp - ODn) × 100, where ODt, ODn, and ODp are the absorbance values ​​of the test sample, negative control, and positive control, respectively.

[0065] (3) Whole blood coagulation test

[0066] Add 500 μL of CaCl2 (0.025 M) solution to 5 mL of whole blood. Transfer 100 μL of activated whole blood to each sample and incubate at 37 °C for 0, 3, 6, 9, 12, 15 and 18 min, respectively. At each time point, add 2.5 mL of sterile water to each well and incubate for another 5 min. After gently mixing, transfer 200 μL of the resulting mixture to a 96-well plate and measure the absorbance at 550 nm. Plot and compare the absorbance curves of the samples over time to obtain the whole blood coagulation kinetic curves.

[0067] (4) Plasma recalcification test

[0068] Add 500 μL of PPP to each sample and blank well, incubate at 37°C with shaking for 1 h, then transfer 100 μL of plasma to a 96-well plate. Immediately add 100 μL of CaCl2 (0.05 M) solution to each well except for the negative control (add 100 μL of physiological saline to the negative control). Measure the absorbance at 405 nm, once every 30 s, for a total of 15 min. Plot the absorbance value over time as the plasma recalcification kinetic curve. The plasma recalcification time is the time required to reach half of the maximum absorbance value (H-M time).

[0069] (5) Blood cell adhesion (platelet adhesion, erythrocyte adhesion, whole blood cell adhesion)

[0070] 500 μL of platelet solution, red blood cell solution, and whole blood cells were added to the surface of the material and incubated at 37°C for 2 h. The samples with attached red blood cells were gently washed with physiological saline, fixed with 2.5% glutaraldehyde for 4 h, and dehydrated with a gradient of ethanol (25%, 50%, 75%, 95%, and 100%, each concentration soaked for 15 min). The ethanol was then replaced with tert-butanol, and the samples were freeze-dried. The platelets, red blood cells, and whole blood cells that adhered to the material were observed by FE-SEM, and the number of adhered blood cells observed in 5K magnification fields was counted (three fields were counted for each sample).

[0071] 5. Evaluation of antibacterial properties

[0072] The antibacterial performance was evaluated using a modified method according to GB / T 20944.2-2007. First, the lyophilized short fiber material was placed in a 24-well plate and sterilized by UV irradiation. Then, 0.5 mL of the activated bacterial strain was inoculated into the well plate and incubated statically for 4 h. After incubation, 1.5 mL of LB medium was added and shaken for 12 h. Then, the plate was serially diluted 10-fold with sterile PBS, and 100 μL of the diluted solution was plated. After incubation at 37°C for 12 h, the plate was photographed and the area occupied by the colonies was measured using Image-J. The antibacterial rate was then calculated.

[0073] 6. In vitro cytotoxicity evaluation

[0074] 400 μL of cell suspension (10% FBS, 1% penicillin-dextrose antibody, 89% DMEM, and 1.0*10⁴ L929 mouse fibroblasts) was seeded onto the surface of each sample and cultured in a 5% CO₂ incubator at 37°C. After culturing L929 cells for 1, 3, and 5 days, the cells were washed three times with PBS, and a mixture of 40 μL of CCK-8 and 360 μL of DMEM was added to each well. After incubation for 1 hour, 100 μL of the mixture was transferred to a 96-well plate, and the absorbance at 450 nm was measured to evaluate the cytotoxicity of the materials and compare the effects of different materials on cell proliferation. To observe the viability of L929 cells, the cells were stained with calcium yellow-green fluorescent dye, and their distribution and morphology were observed under a fluorescence microscope.

[0075] 7. Animal in vivo hemostasis experiment

[0076] All animal experiments complied with the institution's animal protection regulations, and the Animal Research Committee of Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, approved all experiments involving animals. Male SD rats weighing 200-250g were used for animal experiments.

[0077] (1) Statistics on overt hemorrhage

[0078] Three animal hemorrhage models were used to evaluate the hemostatic effect of the sponge in vivo: a tail amputation model, a liver injury model, and a rectus abdominis muscle defect model. For each model, SD rats were divided into 5 groups. Rats were anesthetized by intraperitoneal injection of 1.0% sodium pentobarbital (40 mg / kg), and 50% of the rat's length was removed to create a tail amputation hemorrhage model. A liver injury hemorrhage model was created by exposing the liver through an abdominal incision and pricking it with a 20-gauge needle. A rectus abdominis muscle defect hemorrhage model was created by exposing the rectus abdominis muscle through an abdominal incision and using a blade to create a defect approximately 5 mm in diameter and 1 mm deep. For each hemorrhage model, a pre-weighed sponge was placed over the bleeding site until bleeding stopped, and the weight of the material after absorbing blood was measured. The blood loss for each hemorrhage model was calculated based on the weight of the material before and after blood absorption.

[0079] (2) Changes in hemoglobin after trauma

[0080] A rectus abdominis muscle defect hemorrhage model was used to monitor changes in hemoglobin after hemorrhage, thereby comparing the differences in occult blood loss among different groups. In simple terms, rats were anesthetized by intraperitoneal injection of 1.0% sodium pentobarbital (40 mg / kg). An abdominal incision was made to expose the rectus abdominis muscle. A rectus abdominis muscle defect hemorrhage model with a diameter of approximately 5 mm and a depth of 1 mm was created using a scalpel. Different materials were applied to the defect site for hemostasis, and then the skin was sutured. Approximately 200 μL of blood was collected from the tail vein on days 1, 4, and 7 after hemorrhage. EDTA anticoagulant was added, and hemoglobin levels were measured using a Mindray veterinary fully automated blood cell analyzer (BC-2800vet, Shenzhen, China).

[0081] II. Experimental Results and Characterization

[0082] 1. Analysis of the composition and micro / nano structure of tranexamic acid-functionalized cellulose

[0083] First, such as Figure 1 The diagram shown is a schematic representation of the bacterial BNC production process in the experimental method described above in this invention (e.g., Figure 1 (a) and a schematic diagram of the material preparation process (e.g.) Figure 1 Figure b) shows a schematic diagram of three different complexes (i), (ii), and (iii) that can be constructed using different proportions of OBNC and TXA. Figure 1 (c)

[0084] like Figure 2 As shown in Figure a, four different states of OBNC nanofibers prepared according to this invention are included: emulsion (i), gel (ii), sponge (iii), and powder (iv). The emulsion-like OBNC nanofibers exhibit excellent dispersibility; after standing for 7 days, no stratification or deposition was observed. A gel state was obtained by ultrasonic treatment of 1% (w / v) of OBNC nanofibers. This gel state is injectable and malleable, and can be used for hemostasis of irregular wounds. The sponge-like OBNC nanofibers, obtained after freeze-drying, exhibit a super-fluffy sponge-like structure, compressibility, resilience, and strong water absorption, and can be used for hemostasis of common wounds. The powdered OBNC nanofibers have extremely low density and can be sprayed onto wounds to achieve hemostasis.

[0085] Figure 2 b represents the infrared spectrum analysis of the product, showing that TXA is at 1537 cm⁻¹. -1 and 1643cm -1 There is an absorption peak at 1550 cm⁻¹, which is a characteristic absorption peak of amino groups. OBNC-TXA1 shows an absorption peak at 1550 cm⁻¹. -1 and 1643cm -1 There is an absorption peak at 1560 cm⁻¹, and OBNC-TXA2 has an absorption peak at 1560 cm⁻¹. -1 and 1640cm -1 There is an absorption peak at 1539 cm⁻¹, with OBNC-TXA3 showing an absorption peak at 1539 cm⁻¹. -1 and 1635cm -1 There are absorption peaks at the locations, which are characteristic absorption peaks of amide bond I and amide bond II. There is a shift between the three groups of samples, indicating that there is an excess of amino groups in OBNC-TXA3. The excess amino groups in TXA may also react with their own carboxyl groups to form new amide bonds. In OBNC-TXA2, the carboxyl groups on the surface of OBNC are almost completely consumed. In OBNC-TXA1, the amino groups in the added TXA are insufficient, leaving the remaining carboxyl groups on the surface of OBNC. Figure 2c represents the XPS results, including results in the range of 150–600 eV and N1s 392–410 eV. The N1s energy spectrum analysis shows that trace amounts of N were detected in OBNC-TXA1, indicating that only a small amount of TXA was grafted onto OBNC. Significant amounts of N were detected in both OBNC-TXA2 and OBNC-TXA3 samples, indicating that a certain amount of TXA was present on the samples.

[0086] like Figure 2 In the figure, d represents the observation results of field emission scanning electron microscopy at 5000x and 50000x magnification. At 5000x magnification, three-dimensional micron-sized pores were observed in the four sponge-like materials. At 50000x magnification, it was observed that these sponges were composed of nanofibers with uniform diameter and random arrangement. Therefore, the freeze-dried OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3 materials are short-fiber sponges with nanostructures and micron-sized pores.

[0087] 2. In vitro blood compatibility of tranexamic acid-functionalized cellulose—hemolysis rate

[0088] Hemolysis rate reflects the degree of red blood cell rupture after a material comes into contact with blood. For example... Figure 3 As shown in Figure a, 120 μL of red blood cell solution was added to the surfaces of four materials (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3). The materials rapidly and completely absorbed the red blood cell solution. After incubation for 1 hour and the addition of 2 mL of physiological saline, the results were as follows: Figure 3 As shown in Figure b, red blood cells are suspended in physiological saline and centrifuged as follows: Figure 3 As shown in Figure c, unruptured red blood cells settled at the bottom of the EP tube. The supernatant obtained from the four samples A (OBNC), B (OBNC-TXA1), C (OBNC-TXA2), and D (OBNC-TXA3) was colorless and transparent, indicating that there was no obvious red blood cell rupture. After measuring the absorbance, the hemolysis rate of the four short fibers was calculated as follows: Figure 3 As shown in Figure d, the hemolysis rates were approximately -0.08%, -0.34%, 0.11%, and -0.23%, respectively, with no significant differences among them. Although the short fibers rapidly absorbed the red blood cell solution, the hemolysis rates of all four short fibers were below 0.5%, causing almost no red blood cell rupture, demonstrating excellent blood compatibility and meeting the hemolysis rate requirements for Class III medical devices in the ISO 10993-4:2017 international standard. The negative hemolysis rates of the three short fibers OBNC, OBNC-TXA1, and OBNC-TXA3 are because the degree of hemolysis of the short fibers was lower than that of the blank well plate group, exhibiting excellent blood compatibility. The tranexamic acid functionalization process does not affect the blood compatibility of the cellulose.

[0089] 3. Tranexamic acid-functionalized cellulose promotes whole blood coagulation in vitro.

[0090] The whole blood coagulation process reflects the rate at which complex whole blood clotting occurs upon contact with a material. Dynamic whole blood coagulation processes are used to evaluate the in vitro hemostatic performance of short fibers (e.g., Figure 3 As shown in Figure e), the absorbance values ​​of the solutions after contact with blood at 0, 3, 6, 9, 12, 15, and 18 minutes were as follows: Within 18 minutes, the solution in the plate group was red, and the absorbance value remained almost unchanged, indicating that the blood in the plate group hardly formed clots, resulting in red blood cells remaining suspended in the solution. This suggests that the plate group hardly induced coagulation. The solutions in the four short fiber groups (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3) gradually became transparent over time, and the measured absorbance values ​​all decreased over time, indicating that blood clots gradually formed in the OBNC short fibers and tranexamic acid-functionalized short fibers. All three concentrations exhibited procoagulant effects. OBNC achieved complete clotting within 6 minutes, suggesting its clotting time was approximately 6 minutes. Its procoagulant effect was significantly faster than that of short fibers functionalized with tranexamic acid. OBNC-TXA1 and OBNC-TXA2 had clotting times of approximately 12 minutes, while OBNC-TXA3 exceeded 18 minutes. The clotting rate relationship was OBNC > OBNC-TXA1 > OBNC-TXA2 > OBNC-TXA3, indicating that OBNC had the best procoagulant effect. TXA functionalization significantly slowed the clotting rate, and higher TXA concentrations resulted in poorer clotting. Whole blood clotting time reflects the degree of blood coagulation within 18 minutes, thus demonstrating the inhibitory effect of short fibers on overt bleeding, indicating that OBNC has a significantly superior effect in inhibiting overt bleeding.

[0091] Existing medical hemostatic sponges are prepared by electrospinning, with fiber diameters of approximately 683±147 nm. In contrast, the bacterial nanocellulose fibers in this invention have diameters of only tens of nanometers, resulting in a significantly larger surface area for contact with blood. The varying clotting times observed in different experiments do not necessarily demonstrate that the hemostatic sponge reported in other studies has a superior clotting effect compared to the short fibers in this study. This is related to the addition of Ca to whole blood. 2+ The concentration and degree of blood activation are closely related. Moreover, the whole blood coagulation speed of the OBNC-TAX1 short fibers of the present invention is significantly faster than that of commercial gelatin hemostatic sponges, indicating that the short fibers of the present invention have excellent whole blood coagulation effect.

[0092] Clinically used gelatin hemostatic materials are not easily compressed and hardly expand in volume after absorbing water. OBNC, on the other hand, exhibits a super-fluffy sponge-like structure, is compressible, and expands after absorbing water. OBNC and gelatin hemostatic sponges can absorb 37 times and 5.51 times their own weight in water, respectively. OBNC's water absorption is significantly higher than that of gelatin hemostatic sponges, demonstrating an extremely high water absorption rate. The time required for the weight to no longer change during water absorption is the absorption time, which is 27.6 s for OBNC and 162.6 s for gelatin hemostatic sponges. OBNC's high water absorption rate and rapid absorption speed are mainly attributed to its high porosity and specific surface area. BET testing shows that the porosity of OBNC and gelatin hemostatic sponges is 41 μm. 2 / g and 2.262m 2 / g.

[0093] 4. In vitro plasma recalcification kinetics and plasma recalcification time of tranexamic acid-functionalized cellulose

[0094] Plasma recalcification time is a measure of the coagulation reaction after blood cell-free plasma comes into contact with the material, reflecting the degree of activation of the intrinsic coagulation pathway. The plasma recalcification kinetic curve within 15 minutes is shown below. Figure 3 As shown in f, the plasma recalcification time (HM time) was calculated accordingly. Figure 3 As shown in Figure g, the average plasma recalcification times of the four short fibers (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3) were 4.75 min, 6.57 min, 8.24 min, and 7.3 min, respectively. Among them, OBNC had the shortest plasma recalcification time, followed by OBNC-TXA1. There was no significant difference between OBNC-TXA2 and OBNC-TXA3. This indicates that within 15 min of triggering the coagulation reaction, OBNC short fibers induce coagulation very rapidly, while the combination of TXA slows down the early intrinsic coagulation rate.

[0095] 5. Tranexamic acid-functionalized cellulose surface promotes blood cell adhesion and aggregation in vitro.

[0096] Observing the aggregation and deformation of platelets on the surface of a material is helpful in assessing the material's procoagulant effect. In addition, the adhesion and aggregation of red blood cells also facilitates thrombus formation and promotes the coagulation process. Therefore, observing the aggregation of blood cells on the material surface after co-incubation with the material will help determine the material's coagulation properties. The microscopic morphology of platelets adhering to the material surface is as follows: Figure 4 As shown in Figure b (1000x and 5000x), randomly count the platelet count in three images at 5000x magnification. The results are as follows. Figure 4As shown in Figure e, only about 6 platelets were observed on the surface of the OBNC, while the number of platelets on the surfaces of OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3 were approximately 20, 20, and 20, respectively. There was no significant difference among the three, indicating that the TXA composite promotes platelet adhesion and facilitates the coagulation process. Since whole blood coagulation and plasma recalcification time were only tested within 18 minutes and 15 minutes, respectively, while platelet adhesion was measured after 2 hours of co-incubation with the material, the possible reason is that the composite TXA promotes platelet adhesion and aggregation during the incubation period from 18 minutes to 2 hours, thus exerting a procoagulant effect.

[0097] Microscopic morphology of red blood cells adhering to the material surface, such as Figure 4 As shown in Figure c, the results of randomly counting the number of red blood cells under 5000x magnification are as follows: Figure 4 As shown in Figure f, there was no significant difference in the number of erythrocytes on the surfaces of OBNC-TXA2 and OBNC-TXA3, both significantly lower than the number of erythrocytes on the surfaces of OBNC and OBNC-TXA1, indicating that TXA complexation does not significantly promote erythrocyte aggregation. The adhesion results of whole blood cells are shown in Figure f. Figure 4 As shown in d, the results of randomly counting the number of red blood cells under 5000x magnification are as follows: Figure 4 As shown in Figure g, the number of blood cells adhering to the surface of OBNC-TXA1 is significantly higher than that of OBNC, OBNC-TXA2, and OBNC-TXA3. Simultaneously, a large number of aggregated plasma proteins were observed on the surface of OBNC-TXA1, forming thrombi together with blood cells. This is a result of the combined interaction between the material and various blood cells and plasma, indicating that OBNC-TXA1 is more effective in promoting blood coagulation during the 2-hour co-incubation process. Furthermore, the appropriate amount of TXA on the OBNC surface inhibits plasmin activity, thereby promoting fibrin aggregation and further activating the coagulation pathway.

[0098] 6. Evaluation of the antibacterial properties of tranexamic acid-functionalized cellulose

[0099] Escherichia coli and Staphylococcus aureus were co-incubated with four short fibers and chitosan hemostatic powder for 4 hours. After elution, the mixture was plated. The results are as follows: Figure 5 As shown in Figure b, the statistically calculated inhibition rates against the two bacteria are as follows: Figure 5 As shown in Figure d, it can be seen that OBNC short fibers have the best inhibitory effect on Escherichia coli, and the antibacterial rate decreases with the addition of TXA. OBNC and OBNC-TXA1 have the highest inhibitory efficiency against Staphylococcus aureus, which is consistent with the antibacterial trend against Escherichia coli, meaning that the addition of TXA reduces the antibacterial effect of short fibers.

[0100] 7. In vitro cell compatibility of tranexamic acid-functionalized cellulose

[0101] After co-culturing L929 cells with extracts of four short fibers (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3) for 1, 3, and 5 days, live cells were stained with calcein, and the results were observed under a fluorescence microscope as follows: Figure 5 As shown in Figure c. On the first day, only a few live cells were observed in each group, but by the fifth day, the cell spread rate in each group exceeded 90%. The results of the CCK-8 experiment are shown in Figure c. Figure 5 As shown in Figure e, there was no significant difference in absorbance values ​​between the four material groups on the first and third days. The absorbance values ​​of each group increased over time, indicating that none of the four materials were cytotoxic.

[0102] 8. Tranexamic acid-functionalized cellulose reduces overt blood loss in animals.

[0103] Three animal hemorrhage models (tail amputation model, liver hemorrhage model, and rectus abdominis muscle defect hemorrhage model) were used to further study the hemostatic effects of four short fibers (OBNC, OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3), and the blood infiltration during the hemostasis process was recorded (e.g., Figure 6 (a, b, c) were included, and the amount of blood loss during the bleeding process was calculated (e.g., ...). Figure 6 (e, f, g).

[0104] In the rat tail-severance model, the blood loss in the four short fiber groups was approximately 0.0883g, 0.0169g, 0.0797g and 0.133g, respectively. It can be seen that the blood loss in the OBNC-TXA1 group was significantly lower than that in the other groups.

[0105] In the liver injury bleeding model, the blood loss in the four short fiber groups was approximately 0.0425g, 0.0325g, 0.0526g and 0.0947g, respectively. It can be seen that the blood loss in the OBNC-TXA3 group was significantly higher than that in the other three groups, while the blood loss in the OBNC-TXA1 group was significantly lower than that in the other groups.

[0106] In the rectus abdominis muscle ischemia blood loss model, the blood loss in the four short fiber groups was approximately 0.0266g, 0.0244g, 0.0505g, and 0.0504g, respectively. There was no significant difference between the OBNC group and the OBNC-TXA1 group, and no significant difference between the OBNC-TXA2 group and the OBNC-TXA3 group. However, the blood loss in the OBNC group and the OBNC-TXA1 group was significantly lower than that in the OBNC-TXA2 and OBNC-TXA3 groups.

[0107] Furthermore, in a rat tail amputation model, the blood loss of the gelatin hemostatic sponge was approximately 200 mg; in a rat liver injury model, the blood loss of the gelatin hemostatic sponge was approximately 65.5 mg. These results indicate that the short fibers prepared in this invention have a significantly better hemostatic effect in rat bleeding models than clinically used gelatin hemostatic sponges.

[0108] The hemostasis results from the three animal bleeding models described above show that the OBNC-TXA1 short fibers prepared in this invention have the best effect in reducing overt bleeding. Excessive TXA grafting significantly reduces the original coagulation properties of the OBNC short fibers, resulting in a poorer effect in inhibiting overt bleeding. In summary, the hemostatic performance of OBNC-TXA1 short fibers is significantly better than the other three groups, which is consistent with the in vitro coagulation-promoting results. That is, appropriate TXA grafting onto the OBNC surface is beneficial for activating the coagulation process and reducing overt blood loss in the animal bleeding models.

[0109] To further examine the hemostatic properties of the short fibers prepared in this invention, the hemostatic effect of the OBNC-TXA1 short fibers in this invention was compared with that of the hemostatic material in patent document CN 113667706 A. This patent document describes that the tranexamic acid-loaded cross-linked porous starch hemostatic material prepared therein achieved a free bleeding volume of 0.07g within 10 seconds in a mouse tail-dislocation model. In contrast, the OBNC-TXA1 short fiber hemostatic material in this invention resulted in a total bleeding volume of only 0.0169g in a rat tail-dislocation model over 10 minutes. These results demonstrate that the hemostatic effect of the hemostatic material of this invention is excellent, and even over a longer testing period, the bleeding volume using the hemostatic material of this invention remains significantly lower than that of the hemostatic material in CN 113667706 A.

[0110] 9. Analysis of the inhibitory effect of tranexamic acid-functionalized cellulose on occult bleeding

[0111] A rat model of rectus abdominis muscle defect with hemorrhage was established. Macroscopic images of the materials in vivo one week after the model were compared between the control group (hemostasis with gauze) and the group treated with four different short fibers. Figure 6 As shown in Figure d, no obvious hematoma was observed. The change in hemoglobin levels on day four compared to day one is shown in Figure d. Figure 6As shown in Figure i, the hemoglobin changes in the four short fiber groups were approximately -26.7 g / L, 36.3 g / L, 19.7 g / L, and 14.7 g / L, respectively, while the control group's hemoglobin change was 25.7 g / L. Hemoglobin levels were measured on day 7. In the control group, as well as OBNC-TXA1, OBNC-TXA2, and OBNC-TXA3, all hemoglobin levels returned to normal. Therefore, the hemoglobin change on day 7 compared to day 4 is not meaningful. The OBNC-TXA1 group showed the fastest hemoglobin recovery in the first four days. This is partly because this combined short fiber treatment group had significantly less overt blood loss compared to the other two short fiber treatment groups (OBNC-TXA2 and OBNC-TXA3). Figure 6 (g); On the other hand, the appropriate amount of TXA compound inhibits the activity of plasmin near the bleeding site and increases the activity of fibrin (see schematic diagram). Figure 6 As shown in h), it further aggregates and activates the coagulation pathway, especially the coagulation pathway of open capillaries, reducing the amount of occult blood loss. Therefore, after the wound is sutured, the material inhibits occult bleeding in situ around the bleeding tissue of the wound, allowing the hemoglobin value to quickly return to normal.

Claims

1. A method for preparing a hemostatic material that effectively inhibits overt and covert bleeding, characterized in that, Includes the following steps: (1) Colonies were inoculated into a culture medium for fermentation culture. The culture medium consisted of 100 g / L D-fructose, 5 g / L peptone and 3 g / L yeast extract, pH 5.

0. The culture conditions were constant temperature incubation at 30°C for 7 days. A hydrogel-like bacterial cellulose membrane was obtained on the gas-liquid surface. The bacterial cellulose membrane was sheared, homogenized and freeze-dried. The homogenization operation was performed by homogenizing at 15000 rpm for 15 min to obtain bacterial cellulose nanofibers. (2) Bacterial cellulose nanofibers were dispersed and then oxidized to obtain oxidized bacterial cellulose nanofibers. (3) Disperse 0.3g of oxidizing bacterial cellulose nanofibers in 120mL of 0.05M MES buffer, adjust the pH to 5.5, and add 0.25M NaCl, 0.03M NHS and 0.06M EDC in sequence. Then dispense into 3 bottles, and add 10mg of aminomethyl sulfoxide to one of the bottles. The tranexamic acid was stirred at 30°C for 12 hours, and the oxidizing bacterial cellulose nanofiber composite loaded with tranexamic acid was prepared by EDC / NHS reaction, which is the hemostatic material.

2. The preparation method according to claim 1, characterized in that, The colony mentioned in step (1) is *Acetobacter xylinum*.

3. The preparation method according to claim 1, characterized in that, The preparation method of the oxidizing bacterial cellulose nanofibers in step (2) is as follows: the freeze-dried bacterial cellulose nanofibers are redispersed in sodium phosphate buffer, tetramethylpiperidine oxide and NaClO2 are dissolved in the suspension, NaClO is added dropwise to the sodium phosphate buffer solution, and then the suspension is added immediately. The mixture is magnetically stirred at 50°C for 48 hours. After washing and freeze-drying, the oxidizing bacterial cellulose nanofibers are obtained.

4. The preparation method according to claim 1, characterized in that, The EDC / NHS reaction in step (3) includes the following steps: dispersing oxidizing bacterial cellulose nanofibers into MES buffer, adding NaCl, NHS and EDC in sequence, then adding tranexamic acid, and stirring at 30°C for 12 hours to obtain the product.

5. A highly efficient hemostatic material for inhibiting overt and covert bleeding, prepared by the method described in any one of claims 1-4.

6. The hemostatic material according to claim 5, characterized in that, The hemostatic material may be in the form of emulsion, gel, sponge, or powder.

7. The use of the highly effective hemostatic material for inhibiting overt and covert bleeding prepared by the method of any one of claims 1-4, or the highly effective hemostatic material for inhibiting overt and covert bleeding as described in any one of claims 5-6, in the preparation of medical materials for simultaneously inhibiting overt and covert bleeding.

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