Hemostatic material capable of efficiently inhibiting bleeding and promoting wound repair as well as preparation method and application of hemostatic material

Abstract

The invention relates to a hemostatic material capable of efficiently inhibiting bleeding and promoting wound repair as well as a preparation method and application of the hemostatic material. The hemostatic material is prepared from mutant human thrombin and oxidizing bacterial cellulose nano short fibers. The hemostatic material disclosed by the invention can realize dual effects of inhibiting bleeding in vivo and promoting wound repair, greatly improves the wound repair effect on the premise of rapidly inhibiting bleeding, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of medical hemostatic materials, and particularly relates to a hemostatic material capable of efficiently inhibiting bleeding and promoting wound repair, and a preparation method and application thereof. Background Art

[0002] Bleeding control is a problem that has been studied for generations in human history. Uncontrolled massive bleeding is one of the main causes of death. Globally, the number of deaths caused by trauma each year accounts for about 10% of the total number of deaths, of which 40% of the deaths of trauma patients are caused by massive bleeding; more than 4 million people die after surgery each year worldwide, and postoperative complications (such as bleeding and infection) are the main causes of postoperative death; in modern wars, 75% of casualties are caused by explosive fragments and gunshot wounds, and bleeding is the main cause of death from battlefield trauma. Therefore, achieving rapid and effective hemostasis is a key factor in improving the survival rate of patients in trauma, surgery, and war. In addition to rapid hemostasis, wound healing is a slow and complex process, and additional intervention measures are usually required to achieve better treatment effects and reduce the occurrence of wound infection. It can be seen that in the emergency treatment of accidental trauma in daily life, hemostasis during hospital surgery, and the rescue of injured soldiers in war, local rapid and effective hemostasis and healing of wounds are very important.

[0003] Hemostatic materials used for pre-hospital treatment or surgical bleeding control mainly work through three types of hemostatic mechanisms: (1) Physical mechanisms achieve hemostasis through expansion compression, sealing and absorption of fluid to enrich coagulation-related proteins; (2) Chemical mechanisms trigger and accelerate the hemostatic process by using chemical substances with specific structures or properties such as cationic polymers; (3) Biological mechanisms control bleeding by mimicking the structure or function of platelets or coagulation factors and triggering the natural hemostatic process. Hemostatic materials come in various forms, and the most widely used forms include sponges, hydrogels, nanofibers, and particles or powders.

[0004] Hybrid materials or composite materials are gaining more and more attention. They combine the useful properties of different components, avoid certain shortcomings of single materials, involve multiple hemostatic strategies, and activate primary and secondary hemostasis at the same time through synergistic effects to achieve the purpose of reducing bleeding. However, most local hemostatic agents may not work when the blood coagulation mechanism is impaired. For example, Combat Gauze, a non-woven gauze impregnated with kaolin, cannot act as a coagulant to effectively control bleeding by initiating the coagulation cascade reaction when the blood coagulation mechanism is impaired. Therefore, coagulation factors such as thrombin and fibrinogen that act on the downstream of the coagulation cascade reaction are often used alone as hemostatic materials, or combined with other materials to form composite hemostatic materials.

[0005] Thrombin has multiple functions and plays an important role in promoting coagulation and wound healing. (1) Thrombin promotes hemostasis by acting on multiple substrates: hydrolyzing fibrinogen to promote spontaneous non-covalent binding of fibrin monomers; activating coagulation factor XIII to covalently cross-link adjacent fibrin molecules and enhance the structural integrity of the fibrin network; activated thrombin activates fibrinolysis inhibitors, indirectly enhancing clot stability; hydrolyzing coagulation factors XI, VIII, and V, and positive feedback promotes their own generation. (2) Thrombin promotes platelet activation by activating protease-activated receptors on platelets, leading to platelet aggregation, degranulation, and surface expression of procoagulant phospholipids (such as phosphatidylserine). (3) Thrombin can also stimulate endothelial cells, smooth muscle cells, etc., promote cell surface expression and secretion of cell adhesion molecules and growth factors, and promote cytokines (such as α v β3, VEGF, Ang2, etc.) production.

[0006] But for hemostatic materials, thrombin is a double-edged sword. Thrombin is also involved in the anticoagulation process, binding to thrombomodulin, and selectively inactivating activated coagulation factors V and VIII by activating the protein C anticoagulant pathway, inhibiting the coagulation reaction. In clinical work, researchers have found the prothrombin mutant Arg541Trp in a patient with acute mesenteric venous thrombosis and a positive family history of venous thrombosis. Studies have shown that the ability of mutant thrombin to activate the protein C anticoagulant pathway is reduced. In the thrombin generation test, the mutant prothrombin mutant Arg541Trp promotes more thrombin generation under the same conditions, and shows a coagulation-biased activity compared to the wild type. This suggests that for the development of hemostatic materials, mutant thrombin may achieve better hemostatic effects and become a better choice than wild-type thrombin.

[0007] Currently, many thrombin-based drugs / therapeutics are undergoing clinical trials, some of which have been approved by the FDA, but they still have problems in terms of hemostatic effect, biosafety, and production methods. (1) Bovine plasma-derived thrombin is the most common and cheapest active hemostatic preparation used in the United States and is generally considered the "gold standard" for thrombin products. However, as a heterologous plasma thrombin, patients have allergic reactions after use, which may lead to serious immune-mediated coagulation disorders. (2) Human plasma-derived thrombin undergoes a series of purification procedures during the manufacturing process, but there is still a potential risk of viral infection transmission, and the supply of human-derived blood products is limited and may not be able to meet potential demand. (3) Genetically engineered recombinant expression of human thrombin has higher biosafety. In terms of production, recombinant human thrombin is currently mainly produced in mammalian cell engineering strains such as CHO, but this method is costly and has low yields, which limits its application in pharmaceutical companies; the prokaryotic cell system expresses proteins at a low cost and with high yields, but there are difficulties in protein renaturation, folding, and purification, so it has not yet been widely used. (4) Fibrin glue consists of separately packaged human thrombin and human fibrinogen, and is used as a supportive treatment to improve hemostasis during surgery. However, after it enters the blood vessels, it may cause disseminated intravascular coagulation, thromboembolism, or severe allergic reactions; (5) TachoSil is a collagen sponge encapsulated with human thrombin and human fibrinogen, which is used to stop local bleeding in internal organs during surgery. It reacts with blood, saline or other types of body fluids to form a clot, adhere to the tissue surface, and can achieve hemostasis within a few minutes. However, in some cases, patients may experience allergic reactions or hypersensitivity reactions during treatment.

[0008] Patent CN 116271195A discloses a hemostatic material mixed with acidified oxidized regenerated cellulose loaded with thrombin and oxidized nanocellulose, which is obtained by acidifying oxidized regenerated cellulose with dilute hydrochloric acid, mixing the acidified oxidized nanocellulose and oxidized nanocellulose loaded with Cu2+ with a thrombin solution, washing and drying, and can inhibit in vivo bleeding in animal models in a short time. However, the mixed hemostatic material provided by the patent has the problem that the effect of inhibiting hemostasis needs to be improved. The hemostatic effect of the mixed hemostatic material used in the rabbit liver wound bleeding model shows that the time for complete hemostasis is 2.8 minutes. At the same time, its biosafety and degradability have not been confirmed, and it does not have the effect of promoting wound repair.

[0009] Patent CN116549742A discloses an anti-adhesion superfine fiber membrane made of organic polymer and a hemostatic material attached to the surface of the anti-adhesion superfine fiber membrane, which is prepared by TEMPO-oxidized cellulose nanofiber, chitosan, thrombin and decellularized liver cell extracellular matrix. The composite hemostatic material has the advantages of hemostasis and anti-inflammation, good biocompatibility, non-toxic degradation products to the human body, and can prevent adhesion. However, the effect of promoting coagulation of the composite hemostatic material provided by the patent is not ideal. The test for in vitro coagulation index determination shows that the in vitro coagulation index when investigating 3min is 10%. Therefore, the thrombin material still has the problem of long bleeding time under the premise of achieving hemostasis and anti-inflammation.

[0010] It can be seen that the current hemostatic materials based on thrombin generally use wild-type thrombin derived from animal / human plasma, which has problems such as unsatisfactory hemostatic effect and potential biosafety risks. Therefore, how to avoid the potential safety hazards brought by animal / human plasma products, while giving full play to the hemostatic and repair effects of thrombin, reduce its anticoagulant effect, improve hemostatic efficiency, reduce the amount of bleeding in patients, and shorten the bleeding time of patients has become a technical problem that needs to be solved urgently. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a hemostatic material that can effectively inhibit bleeding and promote wound repair, and a preparation method and application thereof. On the one hand, it solves the problem that thrombin-based hemostatic materials mainly use animal / human plasma products, thereby avoiding their biosafety risks; on the other hand, it solves the problem that thrombin hemostatic materials have insufficient hemostatic effects. While exerting the hemostatic and repair effects of thrombin, its anticoagulant effect is reduced, thereby improving the hemostatic efficiency.

[0012] The invention provides a hemostatic material capable of effectively inhibiting bleeding and promoting wound repair, which is prepared from mutant human thrombin and oxidized bacterial cellulose nano short fibers.

[0013] Preferably, the hemostatic material is in the form of powder, sponge, gel or emulsion.

[0014] The present invention also provides a method for preparing a hemostatic material that can effectively inhibit bleeding and promote wound repair, comprising the following steps:

[0015] (1) fermenting and culturing bacterial colonies to obtain a hydrogel-like bacterial cellulose film, and then performing shearing, homogenization, freeze drying and oxidation treatment to obtain oxidized bacterial nanocellulose short fibers;

[0016] (2) expressing mutant human prothrombin-2 by transforming and inducing bacteria, and obtaining mutant human thrombin with high hemostatic activity by solubilization, renaturation, activation and purification;

[0017] (3) The mutant human thrombin was mixed with oxidized bacterial cellulose nanofibers, and an EDC / NHS grafting reaction was used to prepare a hemostatic material that effectively inhibits bleeding and promotes wound repair.

[0018] Preferably, the bacterial colony in step (1) is Gluconacetobacter xylinum.

[0019] Preferably, the amino acid sequence of the mutant human prothrombin-2 in step (2) is as shown in SEQ NO.1.

[0020] Preferably, the bacteria in step (2) is Escherichia coli BL21 (DE3).

[0021] Preferably, the solubilization in step (2) comprises resuspending the mutant human prothrombin-2 inclusion bodies to 10 mg / mL, using a solubilization solution containing the following components: 23 mM ammonium acetate, 6 M guanidine hydrochloride, 10 mM EDTA, 30 mM cysteine, pH 9.5.

[0022] Preferably, the renaturation in step (2) comprises diluting the soluble mutant human prothrombin-2 protein to 50-100 μg / mL, and the components of the renaturation solution used are: 50 mM Tris-HCl, 20 mM calcium chloride, 500 mM sodium chloride, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v / v) glycerol and 0.2% (w / v) polyethylene glycol hexadecyl, pH 8.5.

[0023] Preferably, the purification in step (2) comprises dialyzing the renatured mutant human prothrombin-2 solution at 25° C. for 1-2 days, using a dialysate containing 20 mM MES, 100 mM sodium chloride, pH 5.5.

[0024] Preferably, the ratio of mutant human thrombin to oxidized bacterial cellulose nanofibers in step (3) is 1000 IU: 1-10 mg.

[0025] Preferably, the EDC / NHS grafting reaction temperature in step (3) is 4° C., and the reaction time is 10-16 h.

[0026] The present invention also provides an application of a hemostatic material capable of efficiently inhibiting bleeding and promoting wound repair in medical hemostasis.

[0027] The present invention synthesizes bacterial cellulose membrane by using Gluconacetobacter xylinum, further prepares it into nano short fibers, and selectively oxidizes the primary alcohol in the side chain of bacterial nano cellulose to carboxyl group through TEMPO chemical oxidation to obtain degradable oxidized bacterial nano cellulose short fibers; the mutant human prothrombin-2 after expression optimization by Escherichia coli is solubilized by guanidine hydrochloride, renatured by dilution, activated by snake venom and purified to obtain mutant human thrombin with high hemostatic activity. The amino group on the mutant human thrombin forms an amide bond with the carboxyl group in the side chain of oxidized bacterial nano cellulose to construct a sponge hemostatic material that can effectively inhibit bleeding and promote wound repair. It has been experimentally verified that the oxidized nano short fibers provided by the present invention can be prepared in various forms, such as powder, sponge, gel and suspension, to meet the hemostatic needs in different bleeding scenarios. The mutant thrombin-grafted oxidized nano-short fiber hemostatic material has an excellent hemostatic effect and promotes the adhesion of various blood components. In the mouse liver bleeding model, it is beneficial to shorten the bleeding time and reduce the amount of bleeding. At the same time, it has an excellent effect of promoting wound repair and is beneficial to the proliferation of mouse embryonic fibroblasts and human umbilical vein endothelial cells. In the mouse full-thickness skin wound model, it has the effect of significantly promoting wound scab and improving the healing rate. It is an efficient hemostatic material that promotes both hemostasis and wound repair.

[0028] Beneficial Effects

[0029] (1) The oxidized bacterial cellulose nanofibers provided by the present invention have a nanoscale structure, high water absorption rate, and can adhere to and concentrate various blood components, activate coagulation reactions, and inhibit bleeding.

[0030] (2) The mutant human thrombin with high hemostatic activity provided by the present invention has high biosafety and excellent hemostatic effect. In the chromogenic substrate test, the ability to activate protein C is weakened and the anticoagulant ability is impaired. In the in vitro coagulation index test, the BCI index reached at different time points is the lowest. In the rat liver bleeding model, it demonstrates excellent procoagulant function.

[0031] (3) The hemostatic material provided by the present invention has the effect of promoting wound repair, promoting the proliferation of human umbilical vein endothelial cells and mouse embryonic fibroblasts and the secretion of related cytokines. In the rat skin full-thickness wound model, the wound surface was kept dry at different time points, the wound healing rate was improved, and it was completely degraded at 21 days, and the treated wound surface was completely scabbed at 21 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Schematic diagram of the synthesis process of OBNC-recombinant human thrombin and its application in hemostasis and angiogenesis (A). The morphology (B) and water absorption rate (C) of sample 1, sample 2, sample 3 and comparative example 1 before (first row) and after (second row) water absorption. The total pore area (D) of samples 1, 2, 3 and comparative example 1.

[0033] Figure 2 The number of adhered red blood cells (A) and the number of blood cells (B) of sample 1, sample 2, sample 3 and comparative example 1. The CCK-8 test results after platelet adsorption by different materials (C). The morphology of the supernatant after the material was incubated with the red blood cell suspension (D) and the red blood cell hemolysis rate (E). The letters a, b, c, d represent sample 1, 2, 3 and comparative example 1, respectively.

[0034] Figure 3 The concentrations of PF4, FV, ADP and 5-HT (AD) after incubation of sample 1, sample 2, sample 3 and comparative example 1 with PRP were measured by ELISA. 2+ Coagulation process and blood coagulation index (BCI) at 3, 6, 9, and 12 min in the presence (E, F) and absence (G, H). Plasma coagulation after incubation of different materials with PPP (I). Absorbance of APC produced at different time points after incubation with PC solution by S-2366 chromogenic substrate method (J). Letters a, b, c, and d represent samples 1, 2, 3, and comparative example 1, respectively.

[0035] Figure 4 In the rat liver injury model, after being treated with Comparative Example 2, Comparative Example 1, Sample 1, Sample 2, and Sample 3, the gross image (A), bleeding volume (B), and blood infiltration area percentage (C) calculated by Image J.

[0036] Figure 5 The proliferation of HUVECs and MEFs in sample 1, sample 2, and sample 3 at 1, 3, and 5 days was measured by CCK-8 method (A, B), and the concentrations of HIF-1α and VEGF secreted by HUVECs (C, D), and the concentrations of MMP-2 and COL I secreted by MEFs (E, F) were measured by ELISA. The tube formation (G), branch node number (I), and total blood vessel length (J) of HUVECs on matrix gel after treatment of sample 1, sample 2, sample 3, and comparative example 1 for 8 and 10 hours. The cell migration (H) and relative migration rate (K) of HUVECs in the scratch test after treatment of negative control, sample 1, sample 2, sample 3, and comparative example 1 for 0 and 12 hours.

[0037] Figure 6In the rat full-thickness skin defect model, gross images and overlapping images of the wound surface (A) and wound healing rates (B) of Comparative Example 2, Sample 1, Sample 2, and Sample 3 0, 7, 14, and 21 days after treatment.

[0038] Figure 7 HE staining (A) and MASSON staining (B) immunohistochemistry results after treatment for Comparative Example 2, Sample 1, Sample 2, and Sample 3 on days 7, 14, and 21, wound thickness (E) and granulation tissue area (F) on day 7. Immunofluorescence results of CD31 staining (rows 1 and 2) and α-SMA staining (rows 3 and 4) (C), number of new blood vessels (G) and number of mature blood vessels (H) on day 7, type I collagen staining (D) and percentage of type I collagen positive area (I) on days 7, 14, and 21, calculated by Image J. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0040] Example 1

[0041] 1. Test methods and materials

[0042] 1. Construction of mutant human thrombin grafted oxidized bacterial nanocellulose short fibers

[0043] (1) Construction of pET-prothrombin-2 plasmid

[0044] The CDS sequence of human prothrombin gene F2 (NM_000506.5) was retrieved in the NCBI database, and the CDS sequence corresponding to prothrombin-2 was extracted (a total of 924 nucleotides, the sequence is shown in SEQ NO.2). The restriction sites Xbal and Xhol were introduced at the amino and carboxyl ends, respectively, and the wild-type and Arg541Trp mutant human prothrombin-2 gene sequences (amino acid sequence is shown in SEQ NO.1) were connected to the prokaryotic expression vector pET-32a (+), and the recombinant plasmid was transformed into BL21 (DE3) competent Escherichia coli. The transformed bacterial solution was screened in LB medium containing 50 μg / mL ampicillin (Amp+), and the positive colonies were screened and cultured and sent to BGI for sequencing and identification, which was consistent with the constructed sequence.

[0045] (2) Expression and refolding of recombinant human prothrombin-2 protein

[0046] The transformed recombinant plasmid solution was cultured in LB medium containing 50 μg / mL AMP+ at 37°C until OD 600 After reaching 0.5, the mixture was induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 4 h, and then centrifuged at 4000 rpm for 15 min at 4°C to obtain a precipitate. The cells were ultrasonically disrupted for 5 min at 40% power for 1 s on and 1 s off, and inclusion bodies were obtained by centrifugation at 15000 rpm for 15 min at 4°C. Insoluble human prothrombin-2 inclusion bodies were resuspended to 10 mg / mL with an increasing solution (23 mM ammonium acetate, 6 M guanidine hydrochloride, 10 mM EDTA, 30 mM cysteine, pH 9.5), and the inclusion bodies were solubilized for 5 min at 40% power for 1 s on and 1 s off. Use refolding solution (50mMTris-HCl, 20mM calcium chloride, 500mM sodium chloride, 1mM EDTA, 600mM arginine, 1mM cysteine, 0.1mM cystine, 10% (v / v) glycerol and 0.2% (w / v) polyethylene glycol hexadecyl, pH 8.5). Dilute the soluble human prothrombin-2 protein to 50-100μg / mL and let it stand at room temperature overnight. The refolded human prothrombin-2 solution is placed in a dialysis bag with a molecular weight cutoff (MWCO) of 10000Da, placed in a dialysis solution (20mM MES, 100mM sodium chloride, pH 5.5) and dialyzed at 25°C for 1 day to obtain the refolded recombinant human prothrombin-2.

[0047] (3) Activation and purification of recombinant human thrombin protein

[0048] The renatured recombinant human prothrombin-2 solution was filtered through a 0.45 μm filter paper and purified using a HiTrap SP HP strong cation exchange chromatography column (Cytiva, USA). Before loading the sample, the column was equilibrated with 30 mL of buffer A (MES 50 mM, NaCl 100 mM, Benzamidine 2 mM, pH 5.5). After loading, the recombinant protein was pure TM Elution was performed on a protein purification system (Cytiva, USA) using gradient buffer B (MES 50mM, NaCl 1M, Benzamidine 2mM, pH 5.5). The eluted sample containing human prothrombin-2 was collected and concentrated using a 10000MWCO ultrafiltration tube. 0.1U of Taipan snake venom and 5mM calcium chloride were added for activation overnight at 4°C, and repurified by a HiTrap SP HP strong cation exchange chromatography column (Cytiva, USA) to obtain recombinant human thrombin protein. The concentrated human thrombin was placed in a 10000MWCO dialysis bag, dialyzed overnight at 4°C with 1000 times the volume of 1×TBS solution, and frozen at -80°C for later use.

[0049] (4) Identification of recombinant human thrombin protein

[0050] After SDS-PAGE, the purified recombinant protein was transferred to a nitrocellulose membrane for Western Blot identification. The primary antibody was mouse anti-human thrombin monoclonal antibody. The secondary antibody was 1:1000 diluted horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody, and DAB staining was performed.

[0051] (5) Activity determination of recombinant human thrombin protein

[0052] The activity of wild-type and mutant recombinant human thrombin was determined by the S-2238 chromogenic substrate method. Commercial bovine thrombin was diluted with 1×TBS solution to standard solutions with activities of 500.0, 250.0, 125.0 and 62.5 IU / mL. Recombinant human thrombin was diluted with 1×TBS solution at a gradient of 1:20, 1:40, 1:80, and 1:160. The S-2238 chromogenic substrate was diluted to 10 nM. The standard and recombinant thrombin samples were added to a U-bottom 96-well plate, and the substrate S-2238 was added. The absorbance was measured at a wavelength of 405 nm for a total of 5 min, with a time interval of 10 s. The average rate and activity of the thrombin standard were used as the standard curve, and the activity of the recombinant human thrombin was calculated by the measured average rate. The experiments were set up with multiple wells and repeated three times.

[0053] (6) Preparation of oxidized bacterial nanocellulose-recombinant human thrombin

[0054] Preparation of bacterial nanocellulose (BNC) short fibers: Gluconacetobacter xylinum was cultured in a fermentation medium at 30°C for 7 days, and a BNC membrane was obtained at the gas-liquid interface. The BNC membrane was boiled at 80°C for 4 hours in a 1% NaOH solution (w / v), and washed repeatedly with ultrapure water until the pH became neutral. It was sterilized by high pressure at 121°C for 30 minutes, repeated 5 times, to ensure that the endotoxin content was lower than the USP standard. The BNC membrane was then cut into small pieces, treated in a homogenizer at 15,000 rpm for 15 minutes, and freeze-dried to obtain BNC short fibers.

[0055] Synthesis of oxidized bacterial nanocellulose (OBNC)-recombinant human thrombin sponge: 0.1 g of BNC short fibers were resuspended in 30 mL of 0.05 M, pH 6.86 sodium phosphate buffer, and 1.7 mmol NaClO2 and 0.01 mM TEMPO were added. 0.2 mL NaClO was added to 10 mL of 0.05 M, pH 6.86 sodium phosphate buffer, and then immediately mixed with the BNC suspension, stirred continuously at 50 ° C for 48 h, and washed with ultrapure water 3 times, and freeze-dried to obtain OBNC. Finally, OBNC (1%, w / v) was suspended with 200 IU / mL recombinant thrombin solution and stirred at 4 ° C for 12 h. OBNC-recombinant human thrombin sponge was obtained after freeze-drying.

[0056] 2. Performance test of OBNC-recombinant human thrombin

[0057] The morphology of OBNC (sample 1), OBNC-wild-type human thrombin (sample 2), OBNC-mutant human thrombin (sample 3) and collagen sponge (Chuangfu Kangguo Medical Device No. 20163142424) (Comparative Example 1) was recorded and photographed. In order to investigate the water absorption performance of the hemostatic material, the freeze-dried samples 1, 2, 3 and comparative example 1 were weighed, and the weighing weight was set to 0.02 g (W dry =0.02) and then immersed in deionized water. Weighing (W) was performed at fixed time points of 10, 20, 30, 60, 120, 180 and 240 s. wet The time during which the weight of the sponge remains unchanged is recorded as the water absorption time (T). The water absorption formula is: water absorption rate (g / s) = (W wet -W dry ) / T. Use photos to record the morphology after water absorption.

[0058] The chemical structure of the hemostatic materials after freeze-drying was investigated by attenuated total reflection infrared spectroscopy. The morphology of the materials after freezing was observed by field emission scanning electron microscopy (FE-SEM). After freeze-drying, the total pore area of ​​the hemostatic materials was analyzed by accelerated surface area and porosimeter (Micromeritics, USA) and automatic mercury porosimeter (Micromeritics, USA) at 30,000 PSIA. The mechanical tensile properties of different materials were measured by dynamic thermal analysis, and the Young's modulus was calculated based on the stress-strain curve.

[0059] 3. Evaluation of in vitro coagulation performance of OBNC-recombinant human thrombin

[0060] (1) Blood compatibility and red blood cell adhesion test

[0061] Drop 120ul of red blood cell suspension into samples 1, 2, 3, comparative example 1, negative control and positive control (n=3). After incubation at 37°C for 2h, add 2mL of normal saline to the incubation solution of samples 1, 2, 3, comparative example 1 and negative control group, add 2mL of sterile water to the positive control group, and incubate again for 1h. After vortex mixing, centrifuge at 660g for 5min, and observe the morphology of the supernatant. Add 100ul of supernatant to a 96-well plate, measure the absorbance at 500nm, and calculate the hemolysis rate of the four groups. Hemolysis rate calculation formula: Hemolysis rate (%) = (OD T -OD N ) / (OD P -OD N )×100%(OD N OD P are the absorbance values ​​of negative control and positive control, respectively).

[0062] (2) Platelet activation and adhesion test

[0063] 500 μL of platelet-rich plasma (PRP) (n=3) was added to samples 1, 2, 3 and comparative example 1, incubated for 5 min, and then centrifuged at 3000 rpm for 5 min. The contents of platelet factor 4 (PF4), 5-hydroxytryptamine (5-HT), ADP and FV in the supernatant were detected using corresponding enzyme-linked immunosorbent assay (ELISA) kits (ELISA Biotechnology, China).

[0064] 500 μL PRP (n=3) was added to samples 1, 2, 3 and comparative example 1, and after incubation for 2 h, the samples were washed 3 times with saline to remove unbound platelets. The sponges were fixed in 2.5% glutaraldehyde for 4 h, dehydrated with 25%, 50%, 75%, 95%, and 100% ethanol gradients, and dried. Platelet activation and adhesion were observed by FE-SEM.

[0065] (3) Coagulation ability and whole blood cell adhesion test

[0066] Add 100 μL of whole blood (with or without 25 mM CaCl2) to samples 1, 2, 3, comparative example 1 and negative control (blank well of 24-well plate without material) and incubate at 37°C for 0, 3, 6, 9, 12 min. Add 2.5 mL of sterile water to different groups at each time point and let stand for 5 min. After mixing, transfer 200 μL of the mixture to a 96-well plate. Measure the absorbance at 550 nm at different time points, calculate the blood coagulation index (BCI), and simulate the whole blood coagulation kinetic curve. Blood coagulation index (BCI) = OD T / OD N × 100%. The material was cleaned and processed as described in the above hemolysis rate experiment. Finally, the blood cells adhered to the sponge surface were counted and observed using FE-SEM.

[0067] (4) Ability to activate the intrinsic coagulation pathway

[0068] 500 μL of platelet-poor plasma (PPP) was added to samples 1, 2, 3, comparative example 1 and negative control (no material). After incubation at 37°C for 1 hour, 100 μL of CaCl2 (5 mM) was added to 100 μL of suspension of samples 1, 2, 3 and comparative example 1, and 100 μL of saline was added to the negative control, and the samples were transferred to a 96-well plate. The absorbance at 405 nm was measured every 30 seconds for 30 minutes, and the changes in absorbance were recorded to draw a kinetic curve.

[0069] (5) Protein C activation ability

[0070] The APC chromogenic substrate method was used to detect the ability of the material to activate the protein C (PC) anticoagulant pathway. Without adding soluble thrombomodulin (sTM), 150 μL of 1 μM PC (1 mg / mL BSA, 0.1% PEG 8000, 1 mM EDTA, 1×TBS) was added to each group of materials and incubated at 37°C. 20 μL of supernatant was aspirated at 0, 20, 40, 60, and 80 min, and hirudin was added to terminate the reaction. 0.5 mM S-2366 chromogenic substrate was added to the mixed solution, and the absorbance was measured at 504 nm.

[0071] 4. Evaluation of in vivo coagulation performance of OBNC-recombinant human thrombin

[0072] Establishment of rat liver bleeding model: 20 SD rats were randomly divided into 5 groups: gauze group (Comparative Example 2), Comparative Example 1, Sample 1, Sample 2 and Sample 3. The rats were anesthetized by intraperitoneal injection of 3.0% sodium pentobarbital (50 mg / kg). The abdomen was disinfected with alcohol, the abdominal cavity was opened, and the liver was exposed. Liver injury was produced by puncturing 5 times with a No. 7 needle at a distance of 7 mm from the lower edge of the liver. The wound was covered with different pre-weighed materials until the blood coagulated and weighed again. The amount of bleeding in the liver injury model was calculated based on the mass change of the material. At the same time, the sponge morphology before and after blood sucking was recorded, and the percentage of blood sucking area was analyzed. In addition, a liver resection bleeding model was produced 3 mm from the lower edge of the liver, and the bleeding time was recorded by video.

[0073] 5. OBNC-recombinant human thrombin related cell experiments

[0074] (1) Cytocompatibility and cell proliferation assays

[0075] 400 μL of 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution, 89% high-glucose DMEM or endothelial cell culture medium (ECM) were added to samples 1, 2, and 3, respectively, and human umbilical vein endothelial cells (HUVECs) or mouse embryonic fibroblasts (MEFs) passaged 2-4 times were inoculated and cultured in a 37°C incubator with 5% CO2. After 1, 3, and 5 days of co-culture, the cells were washed 3 times with 1×PBS, incubated with a mixture of 40 μL CCK-8 and 360 μL DMEM for 1 hour, and then 100 μL of the suspension was transferred to a 96-well plate, and the absorbance was measured at 450 nm. The toxicity of hemostatic substances to cells was evaluated by cell proliferation.

[0076] (2) Cytokine and protein expression detection

[0077] The corresponding ELISA kits (ELISA Biotechnology, China) were used to detect the expression levels of HIF-1α and VEGF in HUVECs treated with different materials to explore the mechanism by which thrombin promotes endothelial cell proliferation and tubule formation; the expression levels of MMP-2 and collagen I in MEFs were detected to clarify the effect of this substance on fibroblast proliferation and collagen production.

[0078] (3) HUVECs migration ability and tube formation experiment

[0079] The scratch test used HUVECs that were passaged 2-4 times. When the cells grew to 90% in a 6-well plate, a straight line was drawn on the bottom of the plate with a 10μL pipette tip to prepare the scratch model, and the detached cells were washed away with 1×PBS. Different materials were added to the 6-well plate and co-cultured with adherent cells. The scratch width was observed and analyzed at 12 and 24 hours.

[0080] HUVECs that were serially passaged 2-4 times were used for tube formation experiments. 50 μL of Matrigel (Biyuntian, China) thawed at 4°C was injected into the bottom of a pre-cooled 96-well plate and then placed at 37°C for 1 hour to allow the Matrigel to solidify. HUVECs (1×10 5 Each well was filled with different materials and incubated at 37°C. After 4, 6, 8 and 10 hours, angiogenesis was observed and photographed, and the number of tubular structures was counted.

[0081] 6. OBNC-recombinant human thrombin in vivo wound repair experiment

[0082] Twenty male SD rats weighing about 250 g were randomly divided into four groups (Comparative Example 2, Sample 1, Sample 2 and Sample 3). Four full-thickness skin defect models with a diameter of 10 mm were established on the back of each rat and treated with different hemostatic materials. The wound area was recorded on the 0th, 7th, 14th, 21st and 28th days of treatment. The wound healing rate was calculated by the formula: Wound healing rate (%) = (S0-ST ) / S0×100%(S0 represents the wound healing area on day 0, S T =Indicates the wound healing area recorded on the day). Tissues were collected and processed with paraffin sections. HE staining and MASSON staining were used to observe wound inflammation and collagen deposition, respectively. CD31, α-SMA, type I collagen and CK14 immunofluorescence staining were used to observe the new blood vessels, mature blood vessels, type I collagen deposition and epithelialization during wound healing.

[0083] 2. Experimental Results

[0084] 1. Preparation and physicochemical properties of OBNC-recombinant human thrombin short fibers

[0085] Depending on the water content, OBNC staple fibers can be in four different forms: powder, sponge, gel and emulsion. The density of powdered OBNC staple fibers is extremely low and can be directly sprayed on the wound to stop bleeding. After ultrasonic treatment, the OBNC staple fibers with a mass concentration of 1% are in a gelatinous state and can be used for injection hemostasis of irregular wounds. Freeze-drying of OBNC staple fibers with a mass concentration of 1% obtains fluffy sponge-like OBNC staple fibers, which are elastic, compressible, and highly absorbent, and can be used for hemostasis of common wounds. The low-concentration OBNC mixture emulsifies after standing for 7 days, showing good antioxidant properties. The present invention selects a commonly used hemostatic sponge form to evaluate the material, OBNC is sample 1, OBNC-recombinant wild-type human thrombin is sample 2, and OBNC-recombinant mutant human thrombin is sample 3. At the same time, collagen hemostatic sponge (COL) is selected as comparative example 1. COL is made of high-purity type I collagen, has the functions of repair, hemostasis, filling, etc., and is currently a commonly used hemostatic material in clinical practice.

[0086] From the macroscopic morphology, samples 1, 2, 3 and comparative example 1 all present a fluffy, compressible sponge morphology ( Figure 1 B). In the water absorption test, there is no significant difference in the water absorption rate of samples 1, 2, and 3, but they are all significantly higher than those of comparative example 1 ( Figure 1 C).

[0087] The short fibers of samples 1, 2, and 3 have uniform diameters and irregular arrangement, and their porosity and specific surface area are higher than those of COL, which is consistent with the water absorption results ( Figure 1 D). Previous studies have shown that OBNC has nanoscale pores and nanofibers with diameters less than 100nm, which can effectively increase the specific surface area of ​​the material and increase contact with liquids, thereby enhancing the hemostatic function. In addition, in order to break the strong hydrogen bonds between the fibers, the OBNC material was treated with ultrasound. The results showed that the force of ultrasound was not enough to break the β-1,4-glucosidic bonds in the fibers and would not affect the function of OBNC.

[0088] 2. In vitro blood compatibility of OBNC-thrombin short fibers

[0089] As a medical device that comes into direct contact with blood, hemostatic materials need to be tested for blood compatibility to determine whether they can be successfully used in clinical practice. This study used an in vitro model and fresh human blood, and the hemolysis rate index was used to reflect the degree of red blood cell rupture in the blood after contact with the material. The supernatant after incubation and centrifugation of samples 1, 2, 3 and comparative example 1 was colorless and transparent ( Figure 2 D), indicating that neither the OBNC sponge nor the grafted thrombin caused obvious erythrocyte rupture.

[0090] In the 450nm wavelength absorbance test, the hemolysis rates of samples 1, 2, 3 and comparative example 1 were all less than 5%, with no significant difference compared with the negative control ( Figure 2 E). The results show that the OBNC short fiber material can absorb red blood cell suspension and has good blood compatibility. It complies with the ISO 10993-4:2017 international standard for hemolysis rate of Class III medical devices, ensuring that the material has sufficient biosafety before clinical application.

[0091] 3. OBNC-thrombin short fibers promote blood cell adhesion and aggregation in vitro

[0092] Blood is a complex organ, consisting of 55% plasma, 44% red blood cells, and 1% white blood cells and platelets. The ability of hemostatic materials to concentrate and adhere to various components in the blood is closely related to their hemostatic function. As the most abundant cell in the blood, the adhesion and aggregation of red blood cells at the bleeding site is the key to improving the mechanical stability of the clot. The results showed that samples 1, 2, and 3 had the largest number of red blood cells, with an adhesion rate of more than 50%, while the adhesion ability of COL was normal ( Figure 2 A).

[0093] Platelets are one of the most important mediators of hemostasis and thrombosis. After incubation of PRP with the material, the platelet adhesion was observed. The CCK-8 absorbance method was used to quantitatively determine the adhered platelets. There was no significant difference in the absorbance values ​​of samples 2 and 3, but they were higher than those of sample 1 and comparative example 1 ( Figure 2 C), indicating that cross-linking of OBNC with thrombin is more conducive to platelet adhesion.

[0094] In the case of calcium addition, the anticoagulated whole blood was incubated with the material, reflecting the complex relationship between the biomaterial and various components in the blood. The adsorption amount of samples 2 and 3 was significantly higher than that of sample 1 and comparative example 1 ( Figure 2 B). Aggregated platelets, plasma proteins and other substances adhere to the sponge surface together with blood cells. These results show that the OBNC sponge has good absorbability after cross-linking with thrombin, which can enrich various components in the blood, play a role in plugging wounds and controlling bleeding.

[0095] 4. ONNC-recombinant mutant human thrombin short fibers promote platelet activation

[0096] Platelets are very important in the hemostasis process, and their deficiency or dysfunction can lead to various coagulation disorders. Platelets are active secretory cells that respond to external stimuli by activating surface receptors and triggering signaling pathways. Thrombin is one of the most effective agonists for platelet activation. It triggers changes in platelet morphology and provides them with additional binding sites for the coagulation cascade. At the same time, it releases a variety of intracellular components such as inflammatory factors, growth factors, coagulation factors, etc. into the bloodstream, interacting with each other and participating in the hemostasis process.

[0097] The α-granules in platelets store coagulation factors such as FV, ​​FX, FXI and platelet factor 4 (PF4). The present invention selects PF4 and FV concentrations as indicators to observe whether platelets are activated and release α-granules. PF4 can promote platelet aggregation and inhibit anticoagulation by binding to heparin. The results show that compared with comparative example 1, when samples 2 and 3 interacted with platelets, the PF4 concentration increased by about 3.9 times ( Figure 3 A). At the same time, the FV concentrations in samples 2 and 3 increased significantly, reaching 786±13 ng / mL and 768±12 ng / mL, respectively, which was about 5.6 times that of comparative example 1 ( Figure 3 B) The secretion of coagulation factors in platelets accelerates the coagulation reaction. Unlike FV in plasma, platelets secrete semi-activated FV with auxiliary factor activity. After activation by thrombin or factor Xa, FV activity is further enhanced by 2 to 3 times.

[0098] In addition, dense granules are also secreted when platelets are activated and release small molecules such as ADP and 5-HT, which are very important for recruiting platelets and expanding platelet responses to bleeding sites. After incubation of samples 2 and 3 with PRP, the concentrations of ADP and 5-TH increased compared with those of control example 1, with sample 2 and sample 3 increasing by about 3.8 times (31.53±0.49mg / mL, 32.36±1.37mg / mL) and 8.3 times (20.46±1.06mg / mL, 20.13±1.19mg / mL) respectively. Figure 3 C, D), indicating that samples 2 and 3 can induce platelet activation, promote the release of α-granules and dense granules, and promote coagulation.

[0099] OBNC-recombinant mutant human thrombin short fibers directly promote the formation of stable clots

[0100] Plasma recalcification test is often used to evaluate the ability of materials to activate the intrinsic coagulation pathway. In the present invention, samples 1, 2, 3 and comparative example 1 were incubated with PPP. In the absence of calcium, samples 2 and 3 produced stable clots, while sample 1 and comparative example 1 did not produce obvious clots ( Figure 3 I). The results showed that thrombin directly acted on the downstream of the common coagulation pathway to hydrolyze fibrinogen without initiating the intrinsic coagulation pathway from scratch, which reflected that samples 2 and 3 had higher hemostatic efficiency.

[0101] In addition to concentrating blood and adhering blood cells, hemostatic materials can also promote the formation of stable blood clots by activating the coagulation cascade. In vitro, the hemostatic ability of the material can be evaluated by monitoring the changes in the blood coagulation index (BCI) over time. The lower the BCI, the larger the blood clot volume and the stronger the hemostatic ability.

[0102] With the addition of calcium ions, the BCI values ​​of samples 2 and 3 were the lowest at 3 min, indicating that it promoted the formation of blood clots ( Figure 3 E, F). However, there was no significant difference between the two thrombins, which may be due to the strong procoagulant function of thrombin. Most of the blood clots had formed at the first time point (3min), and no difference could be observed.

[0103] Without adding calcium ions, only samples 2 and 3 were able to interact with blood to form stable thrombin, indicating that adding thrombin to the material can directly act on the downstream of the coagulation cascade and activate fibrinogen ( Figure 3 G). At 3, 9, and 12 minutes, the BCI value of sample 3 was the lowest among all sample groups, indicating a faster coagulation speed. At 15 minutes, the BCI value of sample 3 (~6%) was significantly lower than that of samples 1, 2, and comparative example 1, indicating that the mutant human thrombin has a more superior hemostatic performance ( Figure 3 H). The BCI of sample 1, comparative example 1 and negative control group decreased over time, but no obvious blood clot formation was observed.

[0104] 6. Impaired PC activation of OBNC-recombinant mutant human thrombin short fibers

[0105] Thrombin can initiate the anticoagulation pathway by activating PC. In order to obtain a better hemostatic effect, this study used the R541W mutant human thrombin found in clinical thrombosis patients. Previous studies have found that in the presence and absence of sTM, the APC conversion rate of the mutant thrombin at 60 minutes was only about 34% and 17% of that of the wild type. The present invention verified the activation effect of samples 2 and 3 on the PC anticoagulation pathway by the chromogenic substrate method. The results showed that in the absence of sTM, the amount of PC converted to APC by sample 3 after 80 minutes of reaction was only about 23% of that of sample 2 ( Figure 3J). For hemostatic materials, the loss of anticoagulant function makes the mutant thrombin more effective in promoting coagulation. Sample 3 can break through the bottleneck of traditional thrombin in hemostasis and improve the hemostatic efficiency of thrombin.

[0106] 7. OBNC-recombinant mutant human thrombin short fibers reduce blood loss in vivo

[0107] In a rat liver injury model, the hemostatic effects of clinical gauze (Comparative Example 2), Comparative Example 1, and samples 1, 2, and 3 were studied ( Figure 4 A). The blood loss was 25.63, 23.85, 15.86, 9.58, and 5.98 mg respectively ( Figure 4 B), the percentage of blood infiltration area on the surface of different hemostatic materials is 74.20%, 66.10%, 18.00%, 16.80%, 9.10% respectively ( Figure 4 C). The hemostatic effect is ranked as follows: sample 3> sample 2> sample 1> comparative example 1> comparative example 2. In addition, it can be seen from the blood infiltration area on the surface of the material that samples 1, 2, and 3 have better water absorption properties than comparative examples 1 and 2 of the commonly used hemostatic materials in clinical practice. In order to make the hemostatic effect more intuitive, we designed a mouse liver lower edge resection model and used sponges for hemostasis. The video results show that sample 3 can control wound bleeding in the shortest time and has the best hemostatic effect. In vivo experiments show that samples 2 and 3 can shorten bleeding time and reduce bleeding volume more than comparison groups 1 and 2, and the hemostatic effect of sample 3 is better than that of sample 2, which is consistent with the results of in vitro coagulation tests.

[0108] 8. In vitro HUVECs and MEFs cell experiments

[0109] In addition to hemostasis, thrombin also participates in the wound healing process by interacting with a variety of cell surface receptors, such as inducing chemotaxis of inflammatory cells, activating endothelial cells, fibroblasts and smooth muscle cells. The results of the CCK-8 experiment showed that after 5 days of culture, HUVECs and MEFs cells continued to proliferate, indicating that both OBNC and recombinant thrombin were non-cytotoxic and the materials had good cell compatibility. On the 3rd and 5th days, the number of cells in samples 2 and 3 was significantly higher than that in sample 1 ( Figure 5 A, B). Previous studies have shown that the acidic environment generated by the carboxyl groups in OBNCs can inhibit cell proliferation, and the addition of thrombin improves this situation.

[0110] In order to clarify the mechanism of promoting cell proliferation in samples 2 and 3, the cell culture medium of each group was further collected and the related factors were detected. In the supernatant co-cultured with HUVECs, the expression levels of HIF-1α in samples 2 and 3 were 6.22±0.27 and 5.23±0.55 pg / mL, respectively ( Figure 5C), and the expression levels of VEGF were 151.39±15.65 and 157.37±16.17 pg / mL ( Figure 5 D), which were higher than those in the OBNC control group. Studies have shown that thrombin can be used as a non-hypoxic stimulus to increase HIF-1α levels and directly upregulate VEGF expression by activating PAR. In the supernatant of co-culture with MEF, the expression levels of MMP-2 in samples 2 and 3 were 16.15±1.30 and 18.46±18.46 ng / mL, respectively ( Figure 5 E). The expression levels of COL I were 3.36±0.19 and 4.41±0.14 ng / mL ( Figure 5 F), which were higher than those in sample 1. Consistent with previous studies, thrombin exerts its profibrotic function by activating MMP-2, causing fibroblasts to produce more collagen.

[0111] New blood vessels can ensure adequate blood flow and provide nutrients and oxygen for the metabolism of various cells around the wound. An important process of angiogenesis is the migration of endothelial cells. The scratch test was used to study the effects of samples 1, 2, and 3 on the migration ability of HUVECs. The results showed that compared with sample 1, the migration ability of HUVECs in samples 2 and 3 was significantly enhanced after 12 hours of co-culture ( Figure 5 H, K). The results showed that thrombin, as a migration signal, enhanced the migration ability of HUVECs. In addition, we studied the promotion effect of different materials on tubule formation in HUVECs to characterize the angiogenesis process ( Figure 5 G). By counting the number of nodes and total length, we found that samples 2 and 3 promoted tubule formation after 4 to 10 hours of treatment compared with the control group ( Figure 5 I, J).

[0112] 9. OBNC-recombinant human thrombin short fibers promote wound healing in vivo

[0113] The wound healing effect was evaluated using a rat full-thickness wound model. According to the gross morphological results of the wound at 0, 7, 14, and 21 days, no obvious infection was observed after surgery, and the wound area decreased with time ( Figure 6 A). At 7 days, the macroscopic morphology of comparative example 2 and sample 1 was incomplete scab, while the wound surface of samples 2 and 3 showed complete scab and the wound surface was relatively dry. At 14 days, each group was completely crusted. The wound healing area of ​​samples 2 and 3 was significantly larger than that of comparative example 2 and sample 1. At 21 days, the wounds treated with samples 2 and 3 were almost completely healed, while the wounds of comparative example 2 and sample 1 were not completely healed. At each time point, the wound healing rate of thrombin-grafted OBNC sponge was good ( Figure 6 B). Thrombin effectively enhances cell proliferation and promotes wound healing by activating PARs and other pathways, while the sponge absorbs secretions to keep it dry and prevent infection.

[0114] HE and MASSON staining were used to evaluate the degree of wound healing, inflammation and collagen deposition during the healing process. HE staining showed that the length of the unrepaired wound at 0, 7, 14, and 21 days was less than 10 mm, indicating that the skin tissue was in a state of repair ( Figure 7 A). The wounds treated with samples 2 and 3 had larger granulation tissue areas, higher morphological density of fibroblasts and inflammatory cells, richer collagen fibers on the wound surface, and slightly stronger inflammatory responses ( Figure 7 F). The wound thickness of samples 2 and 3 was significantly greater than that of the other groups ( Figure 7 E). The reason may be that thrombin activates fibroblasts to secrete extracellular matrix such as collagen and fibronectin. Collagen is deposited on the wound surface and forms granulation tissue together with new capillaries. The formation of granulation tissue can fill tissue defects, play a barrier role, reduce the invasion of pathogenic microorganisms from the wound surface, reduce the risk of infection, and promote wound healing.

[0115] Angiogenesis is an important process in wound healing and repair, providing sufficient nutrients, oxygen and signals for cell proliferation, migration, deposition and extracellular matrix formation. At 7 days, the level of new blood vessels in samples 1, 2 and 3 was higher than that in comparative example 2 ( Figure 7 C, G). The mature blood vessel levels in samples 2 and 3 were significantly higher than those in other groups ( Figure 7 H). The above results suggest that in the first stage of wound healing, transplantation of thrombin materials can promote the induction and maturation of new blood vessels. This is mainly because the thrombin released by the material enhances the surface expression of endothelial cells and secretes cell adhesion molecules (such as α v β3), while promoting the production of growth factors and cytokines.

[0116] The skin repair process is accompanied by the deposition of collagen. The positive area rate of type I collagen in samples 2 and 3 is significantly higher than that in comparative example 2 and sample 1 ( Figure 7 I). Combining the pathological results and neovascularization staining, it was found that treatments of samples 2 and 3 were beneficial to early angiogenesis, promoting collagen deposition and remodeling, and had a positive effect on tissue repair.

[0117] 10. Degradability and pro-inflammatory ability of OBNC-recombinant human thrombin short fibers

[0118] HE staining results showed that after 1 week, a small amount of inflammatory cells (neutrophils, lymphocytes, etc.) infiltrated in the scars treated with samples 2 and 3 ( Figure 7A), no strong acute inflammatory response was produced. Studies have shown that a certain degree of inflammation is beneficial to revascularization and wound healing. As time goes on, cells gradually penetrate into the material. At week 3, many cells proliferated on the wound surface and recovered well. In MASSON staining, the wound surface was covered with red-stained material on the 7th day, but the material was completely degraded after 3 weeks ( Figure 7 B). The rapid degradation rate of short fibers in vivo may be due to the high specific surface area of ​​nanofibers, which enhances the contact between the material and tissue fluid and accelerates the degradation of the material.

Claims

1. A hemostatic material that efficiently inhibits bleeding and promotes wound repair, characterized in that: It is prepared from mutant human thrombin and oxidized bacterial cellulose nanofibers.

2. The hemostatic material according to claim 1, characterized in that: The hemostatic material is in the form of powder, sponge, gel or emulsion.

3. A method for preparing a hemostatic material that efficiently inhibits bleeding and promotes wound repair, comprising the following steps: (1) fermenting and culturing bacterial colonies to obtain a hydrogel-like bacterial cellulose film, and then performing shearing, homogenization, freeze drying and oxidation treatment to obtain oxidized bacterial nanocellulose short fibers; (2) expressing mutant human prothrombin-2 by transforming and inducing bacteria, and obtaining mutant human thrombin with high hemostatic activity by solubilization, renaturation, activation and purification; (3) The mutant human thrombin was mixed with oxidized bacterial cellulose nanofibers, and an EDC / NHS grafting reaction was used to prepare a hemostatic material that can effectively inhibit bleeding and promote wound repair.

4. The preparation method according to claim 3, characterized in that: The bacterial colony in step (1) is Gluconacetobacter xylinum.

5. The preparation method according to claim 3, characterized in that: The amino acid sequence of the mutant human prothrombin-2 in step (2) is shown in SEQ NO.

1.

6. The preparation method according to claim 3, characterized in that: The bacteria in step (2) are Escherichia coli BL21 (DE3).

7. The preparation method according to claim 3, characterized in that: The ratio of mutant human thrombin to oxidized bacterial cellulose nanofibers in step (3) is 1000 IU: 1-10 mg.

8. The preparation method according to claim 3, characterized in that: The EDC / NHS grafting reaction temperature in step (3) is 4° C., and the reaction time is 10-16 h.

9. Use of the hemostatic material for efficiently inhibiting bleeding and promoting wound repair as claimed in claim 1 in medical hemostasis.

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