A liquid hemostatic foam for use in non-compressible hemorrhage and methods of making and using the same

Abstract

The present application belongs to the technical field of hemostatic materials, and particularly relates to a liquid hemostatic foam for incompressible hemorrhage and a preparation method and application thereof. Air is input into a foaming solution to form a foam structure. The foaming solution is prepared by dissolving gelatin, silk fibroin and a metal ion salt in water. In the foaming solution, the concentration of the gelatin is 2% w / v to 8% w / v, the concentration of the silk fibroin is 2% w / v to 8% w / v, and the concentration of the metal ion is 0.8 g / L to 4.0 g / L. The metal ion is calcium ion or ferrous ion. The hemostatic foam has high mechanical strength and good 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 application belongs to the technical field of hemostatic materials, and particularly relates to a liquid hemostatic foam for incompressible hemorrhage and a preparation method and application thereof. BACKGROUND

[0002] In disaster and emergency situations, a large amount of blood loss caused by injury is the main cause of pre-hospital death. Patients need to be operated on quickly, but due to the lack of on-site medical conditions, it may lead to the wounded being unable to receive effective treatment. Therefore, in order to minimize early deaths, it is necessary to intervene and stop bleeding quickly after injury.

[0003] In traumatic hemorrhage, incompressible hemorrhage injury is particularly difficult to diagnose and treat in time, and is still a worldwide problem. For incompressible hemorrhage, so far, blood product transfusion is still the most commonly used method for treating incompressible hemorrhage in clinical practice, but blood products have very high storage requirements, which are difficult to meet the application needs in large-scale wars or disasters. When blood transfusion cannot control bleeding, open surgery or vascular intervention and other means need to be used for intervention. Such methods not only have strict requirements for the professional level of operators, but also are extremely strict for the cleanliness of the environment, and it is difficult to carry out in pre-hospital treatment. In order to meet the clinical needs, various hemostatic devices and drugs have emerged. Various products that have been put on the market have their own advantages, although they have solved some clinical problems, but still have some limitations, which need to be further improved.

[0004] Specifically, the limitations include the following: for incompressible hemorrhage, at present, hemostatic gauze or sponge tamponade is mainly used, and these traditional materials are difficult to reach the deep wound site, in addition, these materials need to be removed by secondary surgery, which will require additional surgical operation process, and there is a risk of secondary bleeding caused by the destruction of the blood clots combined with the materials. As a new type of hemostatic material, injectable gel often needs to determine the position of the bleeding point for hemostatic operation. At the same time, the strong adhesion of hydrogel to tissues can easily cause in vivo tissue adhesion and difficulty in removal and repositioning.

[0005] Unlike morphologically fixed sponges or injectable hydrogels, sprayable liquid foam can cover a larger area with a smaller volume, and can play a large-area hemostatic role in the case of uncertain bleeding points, so as to achieve effective hemostasis of 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 can ensure the potential risks brought by subsequent secondary surgical treatment of the wounded.

[0006] However, when facing high pressure blood flow from incompressible bleeding, the foam material is easily washed away or the structure is destroyed, so it is difficult to achieve sustained 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, surfactants such as sodium dodecyl sulfate (SDS) are usually added to further stabilize the structure of the foam. However, these additives are usually biotoxic. SUMMARY

[0007] To solve the above technical problems, the present application provides a liquid hemostatic foam for incompressible bleeding and a preparation method thereof, the hemostatic foam of the present application has high mechanical strength, good stability, does not contain toxic ingredients, can quickly stop bleeding, and does not conflict with subsequent wound covering treatment.

[0008] The present application is realized by the following technical solutions.

[0009] The present application provides a liquid hemostatic foam for incompressible bleeding, air is input into a foaming solution to form a foam structure;

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

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

[0012] The present application constructs an injectable hemostatic liquid foam system based on gelatin (Gel) and silk fibroin (SF)-Ca 2+ / Fe 2+ complexes, in which metal-protein coordination bonds form complexes. Gelatin is a mixture of various amino acids obtained by hydrolysis of collagen, which can accelerate hemostasis by enriching platelets and concentrating clotting factors. Silk fibroin is a natural biological protein extracted from silk, which can form a stable foam structure by complexing with metal ions, and the two materials can also synergistically promote hemostasis by absorbing blood and promoting coagulation. Among these metal ions, Ca 2+ and Fe 2+ are closely related to the human coagulation mechanism. Ca 2+ acts as a clotting factor IV in the coagulation cascade, with the function of accelerating fibrin clot formation and promoting platelet contraction to accelerate coagulation. Fe 2+ is an important component of hemoglobin, which can quickly flocculate blood to help hemostasis. 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 0.1-0.5:1.

[0015] Preferably, the foam structure is distributed with air bubbles, and the average size of the air bubbles is 50-80 μm, in which size range the foam has optimal mechanical properties and maintenance time.

[0016] The present application provides a preparation method of liquid hemostatic foam for non-compressible hemorrhage, comprising the following steps:

[0017] The foaming solution is prepared by using water as solvent, and gelatin, silk fibroin and metal ion salt as raw materials.

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

[0019] Preferably, the foam structure is formed by using a foaming device, wherein the foaming device comprises 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 by a three-way pipe.

[0020] Preferably, the method for using the foaming device is as follows: air is extracted by the first syringe, the foaming solution is extracted by the second syringe, the three-way pipe is adjusted to make the first syringe and the second syringe communicate, the foaming solution and the air are mixed, and then the first syringe or the second syringe is repeatedly pushed and pulled to make the foaming solution and the air circulate in the pipe of the first syringe or the second syringe, and finally the liquid foam is generated.

[0021] Preferably, the first syringe or the second syringe is repeatedly pushed and pulled for at least 20 times, and more preferably for 20-30 times, so that the air and the foaming solution are sufficiently mixed, and the mechanical strength and stability of the foam are improved.

[0022] Preferably, the foaming solution is prepared by the following method: gelatin and silk fibroin are dissolved in water, and stirred and dissolved at 60°C to prepare a precursor solution, and then a metal ion salt is added to the precursor solution and stirred uniformly to form the foaming solution.

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

[0024] The present application provides the use of the above-mentioned liquid hemostatic foam for non-compressible hemorrhage in the preparation of a non-compressible hemorrhage hemostatic material.

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

[0026] The present application inputs air into a foaming solution 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). The metal-protein complex formed in the liquid hemostatic foam of the present application has the characteristics of a dynamic adjustable coordination bond, can be used to support the foam structure, and acts as a surfactant to achieve the preparation of a foam with good stability and high mechanical strength, and avoid damage to human tissues and organs caused by toxic surfactants.

[0027] Wherein, both gelatin and silk fibroin are FDA-approved and can be used as raw materials for pharmaceutical products. Gelatin is a mixture extracted and purified from animal tissues rich in collagen, which has a unique adhesion motif that can effectively promote the adhesion of hydrogels to the surface of human tissues, organs, etc. When gelatin is dissolved in water to form a gel, it has a rich porous structure that can quickly absorb blood spilled at the wound site, enrich coagulation factors, and accelerate wound hemostasis. Silk fibroin is a natural high molecular weight fibroin extracted mainly 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, promoting the hemostasis process. In addition, it also directly activates coagulation factors or platelets to start the coagulation system. In addition to the hemostatic properties, silk fibroin also supports the growth of keratinocytes and fibroblasts, making it very beneficial for the repair and regeneration of vascular, muscle and other wound tissues. 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, has the function of accelerating fibrin clot formation and promoting platelet contraction to accelerate coagulation. Fe 2+ Is an important component of hemoglobin that can quickly flocculate blood to help hemostasis. In addition, Fe 2+ Can regulate processes such as smooth muscle cell proliferation. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The foaming device structure used in the present application is shown in the figure.

[0029] Figure 2 The photos of the foaming solution before and after foaming in Example 1 of the present application are shown.

[0030] Figure 3 The foaming stability and strength test diagram of the foam is shown. Among them, A is the photo of the foam injected into the EP tube and inverted, B is the photo of the 15ml EP tube opening sealed with foam filled with deionized water, C is the photo of the EP tube in B turned over.

[0031] Figure 4 The morphology of each group of foam samples under optical microscope and the corresponding foam diameter statistical data.

[0032] Figure 5 The foam rheological property test chart. Wherein, A is the experiment chart of exploring the rheological property of the foam by the rheometer and the self-recovery ability under external pressure, B is the G' and G" value change chart of the foam in the cyclic strain shear experiment, C is the curve chart of the foam viscosity changing with the shear rate.

[0033] Figure 6 The foam material biocompatibility test chart. Wherein, A is the hemolysis photo of each group of foams after incubation at 37℃ for 1h, B is the corresponding hemolysis rate calculation value of A, C is the live / dead staining chart, and D is the corresponding cell survival rate quantification data of C chart.

[0034] Figure 7 The foam material in vitro coagulation performance test chart. Wherein, A is the whole blood coagulation test chart, B is the corresponding test data chart of A, C is the in vitro coagulation index test chart, and D is the corresponding test data chart of C.

[0035] Figure 8 The foam material hemostasis test in the rat liver scratch hemostasis model. Wherein, A is the rat liver scratch hemostasis model test and staining chart, B is the rat liver scratch hemostasis model schematic diagram, C is the quantification comparison data of blood loss, hemostasis time and inflammatory factors.

[0036] Figure 9 The rat femoral artery puncture experiment and Bama miniature pig abdominal superficial artery bleeding model test chart. Wherein, A is the rat femoral artery puncture experiment process chart, B is the rat femoral artery puncture experiment schematic diagram, C is the bleeding amount and hemostasis time data in A, and D is the abdominal superficial artery bleeding model test using Bama miniature pig. DETAILED DESCRIPTION

[0037] In order to enable the technical personnel in the art to better understand the technical solutions of the present application, the present application will be further described below in conjunction with specific embodiments and drawings, but the embodiments are not as a limitation on the present application. The experimental methods and detection methods described in the following embodiments are all conventional methods unless otherwise specified; and the reagents and materials described are all commercially available unless otherwise specified.

[0038] The present application provides a liquid hemostatic foam for incompressible bleeding, air is input in a foaming solution to form a foam structure.

[0039] The foaming solution is prepared by dissolving gelatin, silk fibroin and metal ion salt in water, and in the foaming solution, a complex is formed through metal-protein coordination bond, the concentration of gelatin is 2% w / v-8% w / v, the concentration of silk fibroin is 2% w / v-8% w / v, and the concentration of metal ion salt is 0.8 g / L-4.0 g / L.

[0040] The metal-protein complex has dynamic adjustable coordination bond characteristics, can be used to support the foam structure and act as a surfactant, so that the foam with good stability and high mechanical strength is prepared, and damage of toxic surfactant to human tissues and organs is avoided.

[0041] The preparation method of the liquid hemostatic foam for incompressible hemorrhage comprises the following steps:

[0042] The foaming solution is prepared by dissolving gelatin, silk fibroin and metal ion salt in water, and in the foaming solution, a complex is formed through metal-protein coordination bond, the concentration of gelatin is 2% w / v-8% w / v, the concentration of silk fibroin is 2% w / v-8% w / v, and the concentration of metal ion salt is 0.8 g / L-4.0 g / L.

[0043] The foaming solution is mixed with air to form a foam structure, and the volume ratio of the foaming solution to air is 0.1-0.5:1.

[0044] Preferably, the foam structure is formed by using a foaming device, as shown in the figure, the foaming device comprises 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. Figure 1

[0045] Preferably, the method for forming the foam structure by using the foaming device is as follows: air 4 is extracted by using the first syringe 1, the foaming solution 3 is extracted by using the second syringe 2, the three-way pipe 5 is adjusted to make the first syringe 1 and the second syringe 2 communicate, the foaming solution 3 and the air 4 are mixed, then the first syringe 1 or the second syringe 2 is repeatedly pushed and pulled to make the foaming solution 3 and the air 4 circulate in the pipe of the first syringe 1 or the second syringe 2, and finally the liquid foam is generated. The first syringe 1 or the second syringe 2 is repeatedly pushed and pulled at least 20 times, and preferably, the first syringe 1 or the second syringe 2 is repeatedly pushed and pulled for 20-30 times. The first syringe 1 and the second syringe 2 are polyethylene syringes.

[0046] The foaming solution is prepared by dissolving gelatin, silk fibroin and metal ion salt in water, and in the foaming solution, a complex is formed through metal-protein coordination bond, the concentration of gelatin is 2% w / v-8% w / v, the concentration of silk fibroin is 2% w / v-8% w / v, and the concentration of metal ion salt is 0.8 g / L-4.0 g / L.

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

[0048] ​The contents of the present application are specifically illustrated below by the following examples and comparative examples.

[0049] Comparative Example 1

[0050] (1) Preparation of foaming solution

[0051] Gelatin was dissolved in deionized water and stirred in a water bath at 60°C for 1 h to prepare a precursor solution. Subsequently, CaSO4was added to the precursor solution and stirred for another 30 min to obtain a foaming solution. In the foaming solution, the concentration of gelatin was 6% w / v and the concentration of CaSO4was 2.0 g / L.

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

[0053] The foaming device included a first syringe 1 for containing air 4 and a second syringe 2 for containing the foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 were connected by a three-way tube 5. The first syringe 1 and the second syringe 2 were 20 ml polyethylene syringes, and the three-way tube 5 was a silicone tube. The tips of the two polyethylene syringes were connected by the silicone tube to form a structure, in which the first syringe 1 was used to contain air 4 and the second syringe 2 was used to contain the foaming solution 3. The ratio of the foaming solution to air was set at 1:4. By repeatedly pushing and pulling the syringes 20 times, the foaming solution and the gas circulated in the tube, and finally liquid foam was generated, which was recorded as Fy1.

[0054] Comparative Example 2

[0055] (1) Preparation of foaming solution

[0056] Gelatin was dissolved in deionized water and stirred in a water bath at 60°C for 1 h to prepare a precursor solution. Subsequently, FeSO4was added to the precursor solution and stirred for another 30 min to obtain a foaming solution. In the foaming solution, the concentration of gelatin was 6% w / v and the concentration of FeSO4was 2.0 g / L.

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

[0058] The foaming device included a first syringe 1 for containing air 4 and a second syringe 2 for containing the foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 were connected by a three-way tube 5. The first syringe 1 and the second syringe 2 were 20 ml polyethylene syringes, and the three-way tube 5 was a silicone tube. The tips of the two polyethylene syringes were connected by the silicone tube to form a structure, in which the first syringe 1 was used to contain air 4 and the second syringe 2 was used to contain the foaming solution 3. The ratio of the foaming solution to air was set at 1:4. By repeatedly pushing and pulling the syringes 20 times, the foaming solution and the gas circulated in the tube, and finally liquid foam was generated, which was recorded as Fy2.

[0059] Example 1

[0060] (1) Preparation of foaming solution

[0061] Gelatin and silk fibroin powder were dissolved in deionized water, stirred in water bath at 60°C for 1 h to prepare precursor solution. Then, CaS04was added to the precursor solution and stirred for another 30 min to obtain foaming solution. Among them, the concentration of gelatin was 6% w / v, the concentration of silk fibroin was 2% w / v, and the concentration of CaS04was 2.0 g / L.

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

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

[0064] Example 2

[0065] (1) Preparation of foaming solution

[0066] Gelatin and silk fibroin powder were dissolved in deionized water, stirred in water bath at 60°C for 1 h to prepare precursor solution. Then, CaS04was added to the precursor solution and stirred for another 30 min to obtain foaming solution. Among them, the concentration of gelatin was 6% w / v, the concentration of silk fibroin was 2% w / v, and the concentration of CaS04was 2.0 g / L.

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

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

[0069] Example 3

[0070] (1) Preparation of foaming solution

[0071] Gelatin and silk fibroin powder were dissolved in deionized water, stirred in water bath at 60°C for 1 h to prepare precursor solution. Then, CaS04was added to the precursor solution and stirred for another 30 min to obtain foaming solution. Among them, the concentration of gelatin was 8% w / v, the concentration of silk fibroin was 8% w / v, and the concentration of CaS04was 4.0 g / L.

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

[0073] The foaming device included a first syringe 1 and a second syringe 2, the first syringe 1 was used to contain air 4, and the second syringe 2 was used to contain foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 were connected through a three-way tube 5. The first syringe 1 and the second syringe 2 were 20 ml polyethylene syringes, the three-way tube 5 was a silica gel tube, and the tips of the two polyethylene syringes were connected to form a silica gel tube, the first syringe 1 was used to contain air 4, and the second syringe 2 was used to contain foaming solution 3. The ratio of foaming solution to air was set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and the gas circulated in the tube, and finally the liquid foam was generated.

[0074] Example 4

[0075] (1) Preparation of foaming solution

[0076] Gelatin and silk fibroin powder were dissolved in deionized water, stirred in water bath at 60°C for 1 h to prepare precursor solution. Then, CaS04was added to the precursor solution and stirred for another 30 min to obtain foaming solution. Among them, the concentration of gelatin was 8% w / v, the concentration of silk fibroin was 8% w / v, and the concentration of CaS04was 4.0 g / L.

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

[0078] The foaming device included a first syringe 1 and a second syringe 2, the first syringe 1 was used to contain air 4, and the second syringe 2 was used to contain foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 were connected through a three-way tube 5. The first syringe 1 and the second syringe 2 were 20 ml polyethylene syringes, the three-way tube 5 was a silica gel tube, and the tips of the two polyethylene syringes were connected to form a silica gel tube, the first syringe 1 was used to contain air 4, and the second syringe 2 was used to contain foaming solution 3. The ratio of foaming solution to air was set at 1:4. By repeatedly pushing and pulling the syringe 20 times, the foaming solution and the gas circulated in the tube, and finally the liquid foam was generated.

[0079] Example 5

[0080] (1) Preparation of foaming solution

[0081] Gelatin and silk fibroin powders were dissolved in deionized water, stirred in water bath at 60℃ for 1h to prepare precursor solution. Then, CaSO4 was added into the precursor solution and stirred for another 30 minutes to obtain foaming solution. In which, the concentration of gelatin was 6% w / v, the concentration of silk fibroin was 2% w / v, and the concentration of CaSO4 was 2.0g / L.

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

[0083] The foaming device included a first syringe 1 for containing air 4 and a second syringe 2 for containing foaming solution 3, and the outlets of the first syringe 1 and the second syringe 2 were connected by a three-way tube 5. The first syringe 1 and the second syringe 2 were both 20ml polyethylene syringes, and the three-way tube 5 was a silicone tube. The tips of the two polyethylene syringes were connected by the silicone tube to form a structure, in which the first syringe 1 was used to contain air 4 and the second syringe 2 was used to contain foaming solution 3. The ratio of foaming solution to air was set at 1:10. By repeatedly pushing and pulling the syringes for 20 times, the foaming solution and the gas were circulated in the tube, and finally the liquid foam was generated.

[0084] The performances of Examples 2 to 5 were similar to that of Example 1. Hereinafter, only the sample prepared in Example 1 was taken as an example, and the samples prepared in Comparative Examples 1 and 2 were taken as controls to perform performance characterization.

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

[0086] Three liquid hemostatic foams with different formulas were prepared, which were Comparative Example 1, Comparative Example 2 and the samples Fy1, Fy2 and Fy3 prepared in Example 1. First, the foaming stability and strength of the foams were characterized, as shown in A of Figure 3 , the foams were respectively injected and loaded into EP tubes and observed by inverting, and the foams could be stably maintained at the bottom of the tubes without flowing and falling 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 volume of the foam hardly changed. As shown in B and C of Figure 3 , the opening of the 15ml EP tube filled with deionized water was sealed with the foam and turned over, and the foam could withstand the water pressure without being damaged and prevent water from flowing out of the tube. This proved that the liquid foam could fully resist the pressure similar to the blood flow impact after foaming and spraying without being damaged, and exhibited excellent mechanical strength of the liquid foam. The samples in B and C were Fy3 samples. The stability of Fy1 and Fy2 foams was also good.

[0087] As Figure 4 shown, the initial bubble size of the three liquid foams was attributed to the difference in adsorption kinetics of proteins and metal ions, and the change in bubble size over time was mainly related to the stability of the bubble boundary. From the distribution of the figure, it can be found that the average bubble size of Fy2 foam is the largest, about 91.34 microns, and there are larger bubbles, which proves that the metal-protein complex has poor stability for the bubble boundary, the bubble coarsens and coalesces quickly, and finally leads to the decline of the overall stability of the foam, and the average bubble size of Fy3 foam is the smallest and most of them are distributed below 50 microns, which is more stable.

[0088] The rheological properties and self-recovery ability of Fy3 sample foam under external pressure were explored by rheometer. Dynamic scanning was performed in the frequency range of 0.1 to 100 Hz to evaluate the storage modulus (G') and loss modulus (G") of Fy3 and gel foam. As shown in A of Figure 5 , both foams showed solid-like characteristics in the linear viscoelastic range, G'>G", almost independent of frequency. Compared with pure gel foam, the elastic modulus of Fy3 foam was significantly improved. Further study was made on the change trend of G' and G" with the increase of stress at a fixed frequency. As shown in B of Figure 5 , at low stress, the modulus of the foam changed little, but it decreased rapidly after exceeding the yield stress, as indicated by the inflection point in the curve. The yield stress of Fy3 foam was about 60 Pa, while that of gel foam was about 10 Pa. This significant difference highlights the enhancement of the overall mechanical strength of the foam due to metal-protein coordination bonds. The foam was sprayed between parallel plates, then compressed to half of the original height and maintained for one minute, after decompression, the foam could recover to its original shape and adhere to the upper plate, indicating that it had compression resistance and self-recovery performance. Figure C shows that the foam further exhibits its self-recovery performance in the cyclic strain shear experiment, with the solid state characteristics of the gel at 10% strain, G' decreases rapidly and G" almost unchanged at 200% deformation, G" represents the change of material loss modulus, that is, the elasticity of the foam decreases and the viscosity changes little at high strain, G' returns to the original state after the strain is changed to 10%, which proves that the reconstruction of the internal metal bond of the foam makes it have self-recovery ability and will not damage the mechanical structure under high external pressure and can recover quickly. Figure D shows that the viscosity of the foam decreases gradually with the increase of shear rate, that is, the shear thinning property, which proves that the foam can be injected from the syringe without damaging the original structure.

[0089] For biomaterials used in vivo, especially those in contact with blood, their biocompatibility must be strictly evaluated to ensure that they do not adversely affect human health. Hemolytic activity was first characterized by a suspension of rat red blood cells in contact with hemostatic foams. As shown in Figure 6 A and B, after 1 h incubation at 37 °C, the hemolysis rate of each group was less than 5%, meeting the requirements of the American Society of Materials. L929 fibroblasts were used as experimental objects. After 1, 3 and 5 days of culture in medium containing foam extract, cck-8 and live / dead kit detection were performed. As shown in Figure 6 The results of live / dead staining in C showed that both cell types showed normal growth, with green spindle-shaped morphology under fluorescence microscopy, and only a small number of dead cells stained red were observed. As shown in Figure 6 As shown in D, the experimental group showed comparable cell viability to the control group.

[0090] The hemostatic performance of the three foams was verified by in vitro blood clotting test (BCT) and in vitro blood clotting index test (BCI). As shown in Figure 7 As shown in A and B, the BCT of all three hemostatic foam groups was significantly lower, with the BCT of Fy3 foam being the shortest at about 45 seconds. The coagulation ability of the material was observed at two time points of 30 seconds and 300 seconds, and the BCI quantified the coagulation rate; a higher BCI indicates a slower coagulation rate, and the BCI dropped to about 40% at 30 s, as shown in Figure 7 As shown in C, under an optical microscope, it was found that a large number of blood cells were tightly adsorbed in the boundary area of the foam, which may be due to the activation of the Ca 2+ ion-activated coagulation cascade and the enrichment effect of gelatin. Therefore, the hemostatic effect of Fy1 and Fy3 foams is better than that of Fy2 foam. The introduction of silk fibroin further enhances the attraction and enrichment of blood cells due to its negative charge, thereby accelerating hemostasis. Therefore, in the above hemostatic experiment, the Fy3 group has the fastest hemostatic speed. Considering all the results, Fy3 foam was selected for subsequent animal experiments.

[0091] As shown in Figure 8As shown in FIG. 3A and FIG. 3B, Fy3 foam achieved shorter hemostasis time and less blood loss in the rat liver scratch hemostasis model. Foam was sprayed onto the wound immediately after hemostasis, and only a small amount of blood seeped into the underlying gauze. Gelatin sponge could quickly absorb blood, but it took a longer time to achieve complete hemostasis. In the control group, the gauze was heavily soaked with blood, and the hemostasis time was longer. The wound healing was observed after 7 days. In the control group and the gelatin sponge group, the liver lobe and the surrounding tissue were all adhered. The liver tissue in the control group repaired more slowly, and the collagen content was less. The gelatin sponge did not completely degrade in the wound and was observed to remain in the stained sections, hindering further wound recovery. The Fy3 foam group completely degraded, no liver adhesion was observed, and blue collagen was observed in the Masson sections, indicating good tissue recovery. The levels of pro-inflammatory cytokines in the blood of each group of rats were further evaluated using an ELISA kit. As shown in FIG. 3C, compared with the other two groups, the TNF-a level of the Fy3 foam group was significantly reduced, while the IL-1b level showed no significant difference. The biodegradation products of the material can stimulate the production of TNF-a 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 proliferative differentiation phase, thereby promoting wound healing. Figure 8 As shown in FIG. 3C, compared with the other two groups, the TNF-a level of the Fy3 foam group was significantly reduced, while the IL-1b level showed no significant difference. The biodegradation products of the material can stimulate the production of TNF-a 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 proliferative differentiation phase, thereby promoting wound healing.

[0092] As shown in FIG. 3D, further femoral artery puncture experiments were conducted on rats. After the femoral artery was punctured with a 26g needle and bleeding was induced, foam was sprayed at the wound site. Fy3 foam effectively stopped femoral artery bleeding and reduced blood loss. After about 2 minutes, the foam was easily removed with warm water or gauze assistance, and stable blood clots were observed to form, and removing the foam did not damage the blood clots. Figure 9 As shown in FIG. 3D, further femoral artery puncture experiments were conducted on rats. After the femoral artery was punctured with a 26g needle and bleeding was induced, foam was sprayed at the wound site. Fy3 foam effectively stopped femoral artery bleeding and reduced blood loss. After about 2 minutes, the foam was easily removed with warm water or gauze assistance, and stable blood clots were observed to form, and removing the foam did not damage the blood clots. Figure 9 As shown in FIG. 3D, further femoral artery puncture experiments were conducted on rats. After the femoral artery was punctured with a 26g needle and bleeding was induced, foam was sprayed at the wound site. Fy3 foam effectively stopped femoral artery bleeding and reduced blood loss. After about 2 minutes, the foam was easily removed with warm water or gauze assistance, and stable blood clots were observed to form, and removing the foam did not damage the blood clots.

[0093] We also tested the superficial epigastric artery bleeding model of Bama miniature pigs, as shown in FIG. 4A. After the foam was sprayed onto the wound immediately after the bleeding, the wound was observed after 2 minutes. During this period, only a small amount of blood flowed from the gap between the foam and the wound, and eventually achieved hemostasis, with no additional blood flowing around the vascular puncture site. Figure 9 As shown in FIG. 4B, after the foam was sprayed onto the wound immediately after the bleeding, the foam was removed after 2 minutes and the wound was observed. During this period, only a small amount of blood flowed from the gap between the foam and the wound, and eventually achieved hemostasis, with no additional blood flowing around the vascular puncture site.

[0094] These experiments demonstrate the excellent hemostatic effect of Fy3 foam, which is due to the high mechanical stability of the foam, which cannot be damaged by blood flow. At the same time, after hemostasis is complete, the hemostatic foam can be quickly removed from the wound site using gauze or warm water, which will not cause secondary bleeding and is more conducive to further treatment around the wound after initial hemostasis, demonstrating the unique advantages of this type of hemostatic material compared to traditional hemostatic materials.

[0095] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A method for preparing a liquid hemostatic foam for non-compressible bleeding, characterized in that, Includes the following steps: Liquid hemostatic foam for non-compressible bleeding involves introducing air into a foaming solution to form a foam structure. The foaming solution is made by dissolving gelatin, silk fibroin and metal ion salt in water. In the foaming solution, a complex 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 salt is 0.8 g / L to 4.0 g / L. The metal ions are calcium ions or ferrous ions; A foaming solution was prepared using water as a solvent and gelatin, silk fibroin, and metal ion salts as raw materials. The foaming solution is mixed with air to form a foam structure; 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 foaming solution (3). The outlets of the first syringe (1) and the second syringe (2) are connected by a three-way tube (5). The method of forming a foam structure using a foaming device is as follows: air (4) is drawn out with a first syringe (1), foaming solution (3) is drawn out with a second syringe (2), the three-way tube (5) is adjusted to connect the first syringe (1) and the second syringe (2) to mix the foaming solution (3) and air (4), and then the first syringe (1) or the second syringe (2) is pushed and pulled repeatedly to make the foaming solution (3) and air (4) circulate in the tube of the first syringe (1) or the second syringe (2) to finally produce liquid foam.

2. The method for preparing liquid hemostatic foam for non-compressible bleeding according to claim 1, characterized in that, The volume ratio of the foaming solution to air is 0.1~0.5:

1.

3. The method for preparing liquid hemostatic foam for non-compressible bleeding according to claim 1, characterized in that, The foam structure contains air bubbles with an average size of 50μm to 80μm.

4. The method for preparing liquid hemostatic foam for non-compressible bleeding according to claim 1, characterized in that, Push or pull the first or second syringe repeatedly at least 20 times.

5. The method for preparing liquid hemostatic foam for non-compressible bleeding according to claim 1, characterized in that, The preparation method of the foaming solution is as follows: first, dissolve gelatin and silk fibroin in water, heat and stir to dissolve, and prepare a precursor solution. Then, add metal ion salt to the precursor solution and stir evenly to form a foaming solution.

6. The application of the method for preparing liquid hemostatic foam for non-compressible bleeding according to claim 1 in the preparation of hemostatic materials for non-compressible bleeding.

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