Liquid hemostatic foam for non-compressible hemorrhage as well as preparation method and application of liquid hemostatic foam

Abstract

The invention belongs to the technical field of hemostatic materials, and particularly relates to liquid hemostatic foam for non-compressible hemorrhage and a preparation method and application thereof.The preparation method includes the steps that air is input into a foaming solution to form a foam structure; the foaming solution is prepared by dissolving gelatin, silk fibroin and metal ion salt in water, and in the foaming solution, the concentration of the gelatin is 2% w / v-8% w / v, the concentration of the silk fibroin is 2% w / v-8% w / v, and the concentration of metal ions is 0.8 g / L-4. 0g / L; the metal ions are calcium ions or ferrous ions. The hemostatic foam disclosed by the invention is high in mechanical strength and good in stability, does not contain toxic components, can quickly stop bleeding, and does not conflict with subsequent wound covering treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of hemostatic materials, and particularly relates to a liquid hemostatic foam for non-compressible bleeding, a preparation method thereof and an application thereof. Background Art

[0002] In disasters and emergencies, massive blood loss caused by injuries is the main cause of pre-hospital death. Patients need rapid surgery, but due to the lack of on-site medical conditions, it may lead to the inability of the wounded to receive effective treatment. Therefore, in order to minimize early death, it is necessary to intervene and stop bleeding promptly after injury.

[0003] In traumatic bleeding, non-compressible bleeding injuries are particularly difficult to diagnose and treat in a timely manner, and it is still a worldwide problem at present. For non-compressible bleeding, so far, blood product transfusion is still the most commonly used method for treating non-compressible bleeding clinically. However, blood products have very high storage requirements and it is difficult to meet the application needs during large-scale wars or disasters. When blood transfusion cannot control bleeding, means such as open surgery or vascular intervention need to be used for intervention. Such methods not only have strict requirements on the professional level of the operator, but also have extremely strict requirements on the cleanliness of the environment, and it is very difficult to carry out during pre-hospital treatment. In order to meet clinical needs, a variety of hemostatic devices and drugs have emerged. Each of the various products on the market has its own advantages. Although some clinical problems have been solved, there are still some limitations and need to be further improved.

[0004] Specifically, the limitations include the following: For non-compressible bleeding, at present, hemostatic gauze or sponge packing is still mainly used. These traditional materials are difficult to reach the deep wound position. In addition, these materials need to be removed by a second operation, which will require an additional surgical operation process and there is a risk of destroying the blood clot combined with the material and causing secondary bleeding. As a new type of hemostatic material, injectable gel often requires the location of the bleeding point to be clarified for hemostasis operation. At the same time, the strong adhesion of hydrogel to tissues easily leads to problems such as tissue adhesion in the body, difficulty in removal and repositioning.

[0005] Different from sponges or injectable hydrogels with fixed shapes, sprayable liquid foams can cover a larger area with a smaller volume, play a role in large-area hemostasis in the case of uncertain bleeding points, and thus achieve effective hemostasis for deep or irregular wounds. Based on this, a degradable liquid foam hemostatic material is prepared, which can not only provide rapid hemostasis during pre-hospital treatment, but also ensure the potential risks brought by the subsequent secondary surgical treatment of the wounded.

[0006] However, when faced with high-pressure blood flow from non-compressible bleeding, the foam material is easily washed away or its structure is damaged, making it difficult to achieve continuous hemostasis. Therefore, it is necessary to further optimize the mechanical strength and stability of the foam. Due to gravity-induced drainage, bubble coalescence or coarsening, foams usually require the addition of surfactants, such as sodium dodecyl sulfate (SDS), to further stabilize their structure. However, these additives usually have biological toxicity. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a liquid hemostatic foam for non-compressible bleeding and its preparation method. The hemostatic foam of the present invention has high mechanical strength, good stability, does not contain toxic components, can quickly stop bleeding, and does not conflict with subsequent wound covering treatments.

[0008] The present invention is specifically realized through the following technical solutions.

[0009] The present invention provides a liquid hemostatic foam for non-compressible bleeding, in which air is introduced into the foaming solution to form a foam structure;

[0010] The foaming solution is made by dissolving gelatin, silk fibroin and metal ion salt in water. In the foaming solution, the gelatin concentration is 2% w / v - 8% w / v, the silk fibroin concentration is 2% w / v - 8% w / v, and the metal ion salt concentration is 0.8 g / L - 4.0 g / L;

[0011] The metal ion is calcium ion or ferrous ion.

[0012] The present invention constructs an injectable hemostatic foam system based on gelatin (Gel) and silk fibroin (SF)-Ca 2+ / Fe 2+ in which metal-protein coordination bonds form complexes. Gelatin is a mixture of various amino acids obtained by hydrolyzing collagen, and can accelerate hemostasis by enriching platelets and concentrating coagulation factors. Silk fibroin is a natural biological protein extracted from silk. In addition to being able to complex with metal ions to form a stable foam structure, these two materials can also synergistically promote hemostasis by absorbing blood and promoting blood coagulation. Among these metal ions, Ca 2+ and Fe 2+ are closely related to the human blood coagulation mechanism. Ca 2+ acts as coagulation factor IV in the coagulation cascade, and has the functions of accelerating the formation of fibrin clots and promoting platelet contraction to accelerate blood coagulation. Fe 2+ is an important component of heme, and can quickly flocculate blood to help stop bleeding. In addition, Fe 2+ can regulate processes such as smooth muscle cell proliferation.

[0013] Preferably, the calcium ions are derived from CaSO4 or CaCl2, and the ferrous ions are derived from FeSO4 or FeCl2.

[0014] Preferably, the volume ratio of the foaming solution to air is 0.1 to 0.5:1.

[0015] Preferably, the foam structure is distributed with air bubbles, and the average size of the air bubbles is 50 μm to 80 μm. Within this size range, the mechanical properties and maintenance time of the foam are optimal.

[0016] The present invention provides a method for preparing a liquid hemostatic foam for non-compressible bleeding, comprising the following steps:

[0017] Using water as a solvent, gelatin, silk fibroin, and metal ion salts as raw materials, a foaming solution is prepared;

[0018] The foaming solution is mixed with air to form a foam structure.

[0019] Preferably, a foaming device is used to form the foam structure. The foaming device includes a first syringe and a second syringe. The first syringe is used to contain air, the second syringe is used to contain the foaming solution, and the outlets of the first syringe and the second syringe are connected through a three-way tube.

[0020] Preferably, the use method of the foaming device is as follows: Draw air with the first syringe, draw the foaming solution with the second syringe, adjust the three-way tube to connect the first syringe and the second syringe, mix the foaming solution and air, and then repeatedly push and pull the first syringe or the second syringe to make the foaming solution and air circulate in the tube of the first syringe or the second syringe, and finally generate a liquid foam.

[0021] Preferably, the first syringe or the second syringe is repeatedly pushed and pulled at least 20 times. More preferably 20 to 30 times. So that the air and the foaming solution are fully mixed to improve the mechanical strength and stability of the foam.

[0022] Preferably, the preparation method of the foaming solution is as follows: First, dissolve gelatin and silk fibroin in water, stir and dissolve at 60 °C to prepare a precursor solution, and then add the metal ion salt to the precursor solution and stir evenly to form a foaming solution.

[0023] Preferably, in the foaming solution, the concentration of gelatin is 2% w / v to 8% w / v, the concentration of silk fibroin is 2% w / v to 8% w / v, and the metal ion concentration is 0.8 g / L to 4.0 g / L; the metal ion is calcium ion or ferrous ion.

[0024] The present invention provides the application of the above-mentioned liquid hemostatic foam for non-compressible bleeding in the preparation of non-compressible bleeding hemostatic materials.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] Air is introduced into the foaming solution in the present invention to form a liquid hemostatic foam, wherein the main components of the foaming solution are gelatin, silk fibroin and metal ions (specifically calcium ion salt or ferrous ion salt). A metal-protein complex is formed in the liquid hemostatic foam of the present invention, which has the characteristics of dynamically adjustable coordination bonds, can be used to support the foam structure, act as a surfactant, so as to realize the preparation of a foam with good stability and high mechanical strength, and avoid the damage of toxic surfactants to human tissues and organs.

[0027] Among them, both gelatin and silk fibroin are approved by the FDA and can be used as raw materials for pharmaceutical products. Gelatin is a mixture extracted and purified from animal tissues rich in collagen. It has unique adhesion motifs by itself, can effectively promote the adhesion of hydrogel to the surfaces of human tissues, organs, etc. Gelatin has a rich porous structure when dissolved in water, can quickly absorb the blood overflowing from the wound, enrich coagulation factors, and accelerate wound hemostasis. Silk fibroin is a natural high molecular fiber protein mainly extracted from silk, which has excellent mechanical properties, low immunogenicity and high cell compatibility. Silk fibroin can adsorb and concentrate essential blood components such as coagulation factors and platelets to promote the hemostasis process. In addition, it can directly activate coagulation factors or platelets to initiate the coagulation system. In addition to its hemostatic properties, silk fibroin also supports the growth of keratinocytes and fibroblasts, making it very beneficial for the repair and regeneration of traumatized tissues such as blood vessels and muscles. Ca 2+ and Fe 2+ Metal ions closely related to the human coagulation mechanism. Ca 2+ Acts as coagulation factor IV in the coagulation cascade, and has the functions of accelerating the formation of fibrin clots and promoting platelet contraction to accelerate coagulation. Fe 2+ Is an important component of heme, can quickly flocculate blood to help with hemostasis. In addition, Fe 2+ Can regulate processes such as smooth muscle cell proliferation. Brief Description of the Drawings

[0028] Figure 1 It is a schematic structural diagram of the foaming device used in the present invention.

[0029] Figure 2 It is a photo of the foaming solution before and after foaming in Example 1 of the present invention.

[0030] Figure 3 It is a test chart of the foaming stability and strength of the foam. Among them, A is a photo of injecting and loading the foam into an EP tube and inverting it, B is a photo of sealing the opening of a 15 ml EP tube filled with deionized water with the foam, and C is a photo of flipping the EP tube in B.

[0031] Figure 4 Morphology diagrams of each group of foam samples under an optical microscope and corresponding statistical data of foam diameters.

[0032] Figure 5 Diagrams for testing the rheological properties of the foam. Among them, A is an experimental diagram for exploring the rheological properties of the foam and its self - recovery ability under external pressure through a rheometer, B is a diagram showing the numerical changes of G’ and G” during the cyclic strain shear experiment of the foam, and C is a curve diagram showing the change of the foam viscosity with the shear rate.

[0033] Figure 6 Diagrams for testing the biocompatibility of the foam material. Among them, A are hemolysis photos of each group after the foam is incubated at 37 °C for 1 h, B are the calculated numerical values of the hemolysis rate corresponding to A, C is a live / dead staining diagram, and D are the quantified cell survival rate data corresponding to diagram C.

[0034] Figure 7 Diagrams for testing the in vitro blood coagulation performance of the foam material. Among them, A is a diagram of the whole - blood coagulation test, B is a diagram of the test data corresponding to A, C is a diagram of the in vitro blood coagulation index test, and D is a diagram of the test data corresponding to C.

[0035] Figure 8 Hemostasis test of the foam material in the rat liver scratch hemostasis model. Among them, A is a test and staining diagram of the rat liver scratch hemostasis model, B is a schematic diagram of the rat liver scratch hemostasis model, and C are the quantified comparison data of blood loss, hemostasis time, and inflammatory factors.

[0036] Figure 9 Test diagrams of the rat femoral artery puncture experiment and the superficial epigastric artery bleeding model of Bama miniature pigs. Among them, A is a process diagram of the rat femoral artery puncture experiment, B is a schematic diagram of the rat femoral artery puncture experiment, C are the blood loss and hemostasis time data in A, and D is a test using the superficial epigastric artery bleeding model of Bama miniature pigs. Detailed implementation manners

[0037] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. However, the specific embodiments cited do not limit the present invention. In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials are all commercially available unless otherwise specified.

[0038] The present invention provides a liquid hemostatic foam for non - compressible bleeding, and air is introduced into the foaming solution to form a foam structure.

[0039] The foaming solution is prepared by dissolving gelatin, silk fibroin and metal ion salts in water. In the foaming solution, complexation is formed through metal-protein coordination bonds. The concentration of gelatin is 2% w / v to 8% w / v, the concentration of silk fibroin is 2% w / v to 8% w / v, and the concentration of metal ion salts is 0.8 g / L to 4.0 g / L. The metal ion is calcium ion or ferrous ion.

[0040] The metal-protein complex has the characteristics of dynamically adjustable coordination bonds, can be used to support the foam structure, act as a surfactant, so as to realize the preparation of foam with good stability and high mechanical strength, and avoid the damage of toxic surfactants to human tissues and organs.

[0041] A preparation method of a liquid hemostatic foam for non-compressible bleeding includes the following steps:

[0042] Using water as a solvent, gelatin, silk fibroin and metal ion salts as raw materials, a foaming solution is prepared;

[0043] The foaming solution is mixed with air to form a foam structure. The volume ratio of the foaming solution to air is 0.1 to 0.5:1. The foam structure is distributed with air bubbles, and the size of the air bubbles is 50 μm to 80 μm.

[0044] Preferably, a foaming device is used to form the foam structure, such as Figure 1 shown, the foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to contain air 4, the second syringe 2 is used to contain the foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 are connected through a three-way pipe 5.

[0045] Preferably, the method of using the foaming device to form the foam structure is: draw air 4 with the first syringe 1, draw the foaming solution 3 with the second syringe 2, adjust the three-way pipe 5 to connect the first syringe 1 and the second syringe 2, mix the foaming solution 3 and air 4, and then repeatedly push and pull the first syringe 1 or the second syringe 2 to make the foaming solution 3 and air 4 circulate in the pipe of the first syringe 1 or the second syringe 2, and finally generate a liquid foam. The first syringe 1 and the second syringe 2 are repeatedly pushed and pulled at least 20 times. Preferably, the repeated push-pull cycle is 20 to 30 times. The first syringe 1 and the second syringe 2 are polyethylene syringes.

[0046] The preparation method of the foaming solution is: first dissolve gelatin and silk fibroin in water, stir and dissolve at 60 °C to prepare a precursor solution, and then add the metal ion salt to the precursor solution and stir evenly to form a foaming solution.

[0047] The blood foam of the present invention has high mechanical strength, good stability, does not contain toxic components, can stop bleeding quickly, and will not conflict with subsequent wound covering treatment.

[0048] The content of the present invention will be specifically described below through the following examples and comparative examples.

[0049] Comparative Example 1

[0050] (1) Preparation of the foaming solution

[0051] Dissolve gelatin in deionized water and stir it in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. In the foaming solution, the concentration of gelatin is 6% w / v, and the concentration of CaSO4 is 2.0 g / L.

[0052] (2) Preparation of the foaming device and setting of the foaming formula:

[0053] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain the foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silicone tube. The tips of the two polyethylene syringes are connected by a silicone tube. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain the foaming solution 3. The ratio of the foaming solution to air is set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced, denoted as Fy1.

[0054] Comparative Example 2

[0055] (1) Preparation of the foaming solution

[0056] Dissolve gelatin in deionized water and stir it in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add FeSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. In the foaming solution, the concentration of gelatin is 6% w / v, and the concentration of FeSO4 is 2.0 g / L.

[0057] (2) Preparation of the foaming device and setting of the foaming formula:

[0058] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain the foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silicone tube. The tips of the two polyethylene syringes are connected by a silicone tube. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain the foaming solution 3. The ratio of the foaming solution to air is set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced, denoted as Fy2.

[0059] Example 1

[0060] (1) Preparation of foaming solution

[0061] Dissolve gelatin and silk fibroin powder in deionized water, and stir in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. Among them, the gelatin concentration is 6% w / v, the silk fibroin concentration is 2% w / v, and the CaSO4 concentration is 2.0 g / L.

[0062] (2) Preparation of foaming device and setting of foaming formula:

[0063] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silica gel tube. The tips of the two polyethylene syringes are connected by a silica gel tube. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain foaming solution 3. The ratio of foaming solution to air is set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced, denoted as Fy3.

[0064] Example 2

[0065] (1) Preparation of foaming solution

[0066] Dissolve gelatin and silk fibroin powder in deionized water, and stir in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. Among them, the gelatin concentration is 2% w / v, the silk fibroin concentration is 6% w / v, and the CaSO4 concentration is 0.8 g / L.

[0067] (2) Preparation of foaming device and setting of foaming formula:

[0068] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silica gel tube. The tips of the two polyethylene syringes are connected by a silica gel tube. The first syringe 1 is used to contain air 4, and the second syringe 2 is used to contain foaming solution 3. The ratio of foaming solution to air is set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced.

[0069] Example 3

[0070] (1) Preparation of foaming solution

[0071] Dissolve gelatin and silk fibroin powder in deionized water, and stir in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. Among them, the gelatin concentration is 8% w / v, the silk fibroin concentration is 8% w / v, and the CaSO4 concentration is 4.0 g / L.

[0072] (2) Preparation of foaming device and setting of foaming formula:

[0073] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silica gel tube. The tips of the two polyethylene syringes are connected by the silica gel tube. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold foaming solution 3. The ratio of the foaming solution to air is set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced.

[0074] Example 4

[0075] (1) Preparation of foaming solution

[0076] Dissolve gelatin and silk fibroin powder in deionized water, and stir in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. Among them, the gelatin concentration is 6% w / v, the silk fibroin concentration is 2% w / v, and the CaSO4 concentration is 2.0 g / L.

[0077] (2) Preparation of foaming device and setting of foaming formula:

[0078] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silica gel tube. The tips of the two polyethylene syringes are connected by the silica gel tube. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold foaming solution 3. The ratio of the foaming solution to air is set at 1:2. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced.

[0079] Example 5

[0080] (1) Preparation of foaming solution

[0081] Dissolve gelatin and silk fibroin powder in deionized water, stir in a water bath at 60 °C for 1 h to prepare a precursor solution. Subsequently, add CaSO4 to the precursor solution and stir for another 30 minutes to obtain a foaming solution. Among them, the gelatin concentration is 6% w / v, the silk fibroin concentration is 2% w / v, and the CaSO4 concentration is 2.0 g / L.

[0082] (2) Preparation of foaming device and setting of foaming formula:

[0083] The foaming device includes a first syringe 1 and a second syringe 2. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold the foaming solution 3. The outlets of the first syringe 1 and the second syringe 2 are connected through a three-way tube 5. The first syringe 1 and the second syringe 2 are both 20 ml polyethylene syringes, and the three-way tube 5 is a silica gel tube. The tips of the two polyethylene syringes are connected by a silica gel tube. The first syringe 1 is used to hold air 4, and the second syringe 2 is used to hold the foaming solution 3. The ratio of the foaming solution to air is set at 1:10. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and gas circulate in the tube, and finally liquid foam is produced.

[0084] The performances of Examples 2 to 5 are similar to those of Example 1. Below, only the samples prepared in Example 1 are taken as examples, and the samples prepared in Comparative Example 1 and Comparative Example 2 are used as controls for performance characterization.

[0085] Figure 2 Photos before and after foaming in Example 1.

[0086] Three kinds of liquid hemostatic foams with different formulas were prepared, namely the samples Fy1, Fy2, and Fy3 prepared in Comparative Example 1, Comparative Example 2, and Example 1 respectively. First, the foaming stability and strength of the foams were characterized. As Figure 3 shown in A, the foams were respectively injected and loaded into EP tubes and inverted for observation. The foams could be stably maintained at the bottom of the tubes without flowing off. Even after 14 days of long-term observation, except that the Fy2 foam turned yellow due to the oxidation of divalent iron to trivalent iron ions, only slight coarsening was observed in each group of foams, and the foam volume hardly changed. As Figure 3 shown in B and C, use the foam to seal the opening of a 15 ml EP tube filled with deionized water and turn it over. The foam can withstand the water pressure without being damaged and prevent water from flowing out of the tube. This proves that the liquid foam can fully resist the pressure similar to blood flow impact without being damaged after foaming and spraying, showing excellent mechanical strength of the liquid foam. The samples in B and C are Fy3 samples. The stabilities of Fy1 and Fy2 foams are also good.

[0087] As Figure 4 shown, the bubble sizes of three liquid foams were observed under an optical microscope and quantified using ImageJ software. The initial bubble sizes of the foams can be attributed to the differences in the adsorption kinetics of proteins and metal ions, while the change in bubble size over time is mainly related to the boundary stability. From the distribution in the figure, it can be found that the Fy2 foam has the largest average bubble size, approximately 91.34 μm on average, and there are larger bubbles, which proves that the metal-protein complex has poor stability at the bubble boundary, and the bubbles quickly coarsen and coalesce, ultimately leading to a decrease in the overall stability of the foam. The Fy3 foam has the smallest average bubble size and most of them are distributed below 50 μm, being more stable.

[0088] The rheological properties of the Fy3 sample foam and its self-recovery ability under external pressure were investigated using a rheometer. Dynamic scans were performed in the frequency range of 0.1 to 100 Hz to evaluate the storage modulus (G') and loss modulus (G") of the Fy3 and gel foams. As Figure 5 shown in A of Figure 5 , both foams exhibit solid-like characteristics within the linear viscoelastic range, G' > G", and are almost independent of frequency. Compared with the pure gelatin foam, the elastic modulus of the Fy3 foam is significantly increased. The variation trends of G' and G" with the increase in stress at a fixed frequency were further studied. As

[0089] shown in B of , at low stress, the modulus of the foam changes little, but it decreases rapidly after exceeding the yield stress, as shown by the inflection point in the curve. The yield stress of the Fy3 foam is approximately 60 Pa, while that of the gel foam is approximately 10 Pa. This significant difference highlights the enhancement of the overall mechanical strength of the foam due to the metal-protein coordination bond. The foam was sprayed between parallel plates, then compressed to half of its initial height and maintained for one minute. After decompression, the foam was able to recover its original shape and adhere to the upper plate, and the foam was not damaged due to extrusion, indicating its compressive resistance and self-recovery performance. Figure C shows that the foam further demonstrated its self-recovery performance in the cyclic strain shear experiment. It has the solid-state characteristics of a gel at 10% strain, G' decreases rapidly while G" changes little at 200% deformation. G" represents the change in the loss modulus of the material, that is, the elasticity of the foam decreases while the viscosity changes little at high strain. After the strain changes back to 10%, G' returns to its original state, which proves the reconstruction of the internal metal bonds in the foam, enabling the foam to have self-recovery ability, not to damage the mechanical structure under high external pressure and to be able to recover quickly. Figure D shows that the viscosity of the foam gradually decreases with the increase in the shear rate, that is, the shear-thinning property, which proves that the foam can be injected from a syringe without damaging the original structure.For biomaterials used in vivo, especially those that come into contact with blood, their biocompatibility must be strictly evaluated to ensure that they do not have an adverse impact on human health. Hemolytic activity was first characterized by a suspension of rat red blood cells in contact with the hemostatic foam. As Figure 6 shown in A and B, after incubation at 37 °C for 1 h, the hemolysis rate of each group was less than 5%, meeting the requirements of the American Society for Testing and Materials. L929 fibroblasts were used as the experimental subjects. CCK-8 and live / dead cell kits were used for detection after culturing for 1, 3, and 5 days in a medium containing foam extract. As Figure 6 shown in the live / dead staining result in C, both cell types showed normal growth, presenting a green spindle shape under a fluorescence microscope, and only a small number of dead cells stained red were observed. As Figure 6 shown in D, the experimental group showed cell viability comparable to that of the control group.

[0090] The hemostatic performance of the three foams was verified by in vitro whole blood clotting test (BCT) and in vitro blood coagulation index test (BCI). As Figure 7 shown in A and B, the BCT of all three hemostatic foam groups was significantly lower, and the BCT of the Fy3 foam was the shortest, approximately 45 seconds. The clotting ability of the materials was observed at two time points of 30 seconds and 300 seconds, and the BCI quantified the clotting rate; a higher BCI indicates a slower clotting rate, and the BCI decreased to about 40% at 30 s, as Figure 7 shown in D. As shown in C, the contact between the foam and blood was observed under an optical microscope, and a large number of blood cells were found to be tightly adsorbed in the foam boundary region, which may be due to the combined effect of Ca 2+ ion activation of the coagulation cascade and the enrichment effect of gelatin. Therefore, the hemostatic effects of the Fy1 and Fy3 foams were better than that of the Fy2 foam. The introduction of silk fibroin further enhanced the attraction and enrichment of blood cells due to its negatively charged characteristics, thus accelerating hemostasis. Therefore, in the above hemostatic experiment, the Fy3 group had the fastest hemostatic speed. Considering all the results, the Fy3 foam was selected for subsequent animal experiments.

[0091] As Figure 8As shown in A and B, in the rat liver scratch hemostasis model, the hemostasis time of Fy3 foam was short and the blood loss was small. Hemostasis occurred immediately after the foam was sprayed on the wound, and only a small amount of blood seeped into the underlying gauze. The gelatin sponge could rapidly absorb blood, but it took a longer time to achieve complete hemostasis. In the control group, the gauze was extensively soaked with blood and the hemostasis time was longer. The wound healing was observed after 7 days. Adhesions occurred between the liver lobes and the surrounding tissues in both the control group and the gelatin sponge group. The liver tissue repair was slower and the collagen content was less in the control group. The gelatin sponge was not completely degraded in the wound and residues were observed in the stained sections, which hindered further wound recovery. The Fy3 foam group was completely degraded, no liver adhesions were observed, and blue collagen was visible in the Masson sections, indicating good tissue recovery. The levels of pro-inflammatory cytokines in the blood of rats in each group were further evaluated using an ELISA kit. As Figure 8 shown in C, compared with the other two groups, the TNF-α level in the Fy3 foam group was significantly reduced, while there was no significant difference in the IL-1β level. The biodegradation products of the materials may stimulate the production of TNF-α and exacerbate the inflammatory response. However, the degradation products of Fy3 foam, due to their excellent biocompatibility and partial inhibition of inflammatory expression, accelerated the transition from the inflammatory phase to the proliferation and differentiation phase, thus promoting wound healing.

[0092] As Figure 9 shown in A and B, the rat femoral artery puncture experiment was further carried out. After puncturing the femoral artery with a 26g needle and inducing bleeding, the foam was sprayed on the wound. Fy3 foam could effectively stop the bleeding of the femoral artery and reduce the blood loss. After about 2 minutes, the foam could be easily removed with the assistance of warm water or gauze, and a stable blood clot was observed to form. Removing the foam did not damage the blood clot. As Figure 9 shown in C, the blood loss and hemostasis time in the gauze and gelatin sponge control groups were higher than those in the Fy3 foam group.

[0093] We also used the superficial epigastric artery bleeding model of Bama mini-pigs for testing. As Figure 9 shown in D, the foam was sprayed onto the wound immediately after puncture bleeding. Since it was difficult to observe the wound condition due to the foam covering the bleeding point, the foam was removed and observed after 2 minutes. During this period, only a small amount of blood flowed out from the gap between the foam and the wound, and hemostasis was finally successfully achieved, with no additional flowing blood around the vascular puncture site.

[0094] These experiments demonstrated the excellent hemostatic effect of Fy3 foam. Due to its high mechanical stability, the overall structure of the foam was not damaged by the blood flow. At the same time, after hemostasis was completed, the hemostatic foam could be quickly removed from the wound by rinsing with gauze or warm water, which neither caused secondary bleeding nor was more conducive to further treatment around the wound after initial hemostasis, demonstrating the unique advantages compared to traditional hemostatic material types.

[0095] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, these modifications and variations are also intended to be included therein.

Claims

1. A liquid hemostatic foam for non-compressible bleeding, characterized in that, Air is introduced into the foaming solution to form a foam structure; The foaming solution is made by dissolving gelatin, silk fibroin and metal ion salts in water. In the foaming solution, a complex is formed through metal-protein coordination bonds. Among them, the concentration of gelatin is 2% w / v to 8% w / v, the concentration of silk fibroin is 2% w / v to 8% w / v, and the concentration of metal ion salts is 0.8 g / L to 4.0 g / L; The metal ion is calcium ion or ferrous ion.

2. The liquid hemostatic foam for non-compressible bleeding according to claim 1, wherein The volume ratio of the foaming solution to air is 0.1 to 0.5:

1.

3. The liquid hemostatic foam for non-compressible bleeding according to claim 1, characterized in that, Bubbles are distributed in the foam structure, and the average size of the bubbles is 50 μm to 80 μm.

4. The preparation method of the liquid hemostatic foam for non-compressible bleeding according to claim 1, wherein, It includes the following steps: Using water as a solvent and gelatin, silk fibroin and metal ion salts as raw materials, a foaming solution is prepared; The foaming solution is mixed with air to form a foam structure.

5. The preparation method according to claim 4, wherein A foaming device is used to form a foam structure. The foaming device includes a first syringe (1) and a second syringe (2). The first syringe (1) is used to hold air (4), and the second syringe (2) is used to hold the foaming solution (3). The outlets of the first syringe (1) and the second syringe (2) are connected through a three-way pipe (5).

6. The preparation method according to claim 5, characterized in that, The method of using the foaming device to form a foam structure is as follows: The first syringe (1) is used to draw air (4), the second syringe (2) is used to draw the foaming solution (3), the three-way pipe (5) is adjusted to connect the first syringe (1) and the second syringe (2), the foaming solution (3) and air (4) are mixed, and then the first syringe (1) or the second syringe (2) is repeatedly pushed and pulled to make the foaming solution (3) and air (4) circulate in the tube of the first syringe (1) or the second syringe (2), and finally liquid foam is produced.

7. The preparation method according to claim 6, characterized in that, The first syringe or the second syringe is repeatedly pushed and pulled at least 20 times.

8. The preparation method according to claim 4, characterized in that, The method for preparing the foaming solution is as follows: First, gelatin and silk fibroin are dissolved in water, heated and stirred to dissolve to prepare a precursor solution, and then the metal ion salt is added to the precursor solution and stirred evenly to form a foaming solution.

9. The preparation method according to claim 4, characterized in that, In the foaming solution, the concentration of gelatin is 2% w / v to 8% w / v, the concentration of silk fibroin is 2% w / v to 8% w / v, and the metal ion concentration is 0.8 g / L to 4.0 g / L; the metal ion is calcium ion or ferrous ion.

10. Use of the liquid hemostatic foam for non-compressible bleeding in the preparation of a hemostatic material for non-compressible bleeding.

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