Collagen composite hemostatic sponge as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical materials, and particularly discloses a collagen composite hemostatic sponge and a preparation method and application thereof.The collagen composite hemostatic sponge is prepared from raw materials including polypeptide, collagen and scaffold protein through self-assembly, in some cases, active matters can be added into the collagen composite hemostatic sponge to further improve various properties of the collagen composite hemostatic sponge. The collagen composite hemostatic sponge provided by the invention avoids the residue of a cross-linking agent in the traditional technology, not only can absorb a large amount of liquid, but also has strong adhesion to platelets and blood cells, is beneficial to the activation of the platelets and the activation and coagulation process of the blood cells, and realizes rapid coagulation; meanwhile, the collagen composite hemostatic sponge provided by the invention also has excellent biocompatibility, is an absorbable hemostatic material, avoids frequent replacement, and reduces the pain of a patient.

Description

Technical Field

[0001] The present application belongs to the technical field of medical materials, and specifically relates to a collagen composite hemostatic sponge and a preparation method and application thereof. Background Art

[0002] As an important part of modern medical treatment, surgical operations often require hemostasis during the operation. For this reason, hemostatic materials are often needed to fill the wound in order to achieve the purpose of rapid hemostasis. Currently, gauze and bandages are still commonly used to control bleeding. However, conventional materials such as gauze and bandages have problems such as excessive permeability, wound adhesion, and microbial entry. At the same time, they are prone to cause discomfort to patients when replaced.

[0003] In order to overcome the problems brought by conventional hemostatic materials in the prior art, hemostatic sponges have been developed. However, in actual applications, it is found that existing hemostatic sponges often have problems such as poor mechanical properties, which can cause the existing hemostatic sponges to collapse after absorbing liquid, resulting in a decrease in hemostatic performance.

[0004] Therefore, researching and developing hemostatic materials with high hemostasis efficiency and excellent biocompatibility to alleviate patients' pain has become an important research direction. Summary of the invention

[0005] The purpose of this application is to solve the deficiencies of the prior art and provide a collagen composite hemostatic sponge and a preparation method and application thereof, specifically adopting the following technical solutions:

[0006] In a first aspect, the present application discloses a collagen composite hemostatic sponge, comprising a first material, wherein the first material is obtained by self-assembly of raw materials including a polypeptide, collagen and a scaffold protein; the mass ratio of the collagen, polypeptide and scaffold protein is (5-50): (2.5-16): (0.5-10).

[0007] Preferably, the mass ratio of collagen, polypeptide and scaffold protein is (10-25):(4-12):(1.5-10).

[0008] In the prior art, a cross-linking agent is often added to collagen to prepare a hemostatic sponge. Although this method is simple and cheap, due to the addition of a cross-linking agent, there is also a technical problem that the collagen structure is destroyed due to the cross-linking agent residue. However, the present application uses scaffold protein, combined with collagen and polypeptide to prepare a collagen composite hemostatic sponge that avoids the use of a cross-linking agent. In addition, its hemostatic ability, liquid absorption capacity and biocompatibility are significantly improved.

[0009] Preferably, the scaffold protein used in the present application is RACK1 protein; the polypeptide used in the present application is Fibronectin Active Fragment Control.

[0010] In some preferred implementations, the collagen composite hemostatic sponge further comprises an active substance; the active substance comprises at least one of rhamnosyl uridine, silymarin, puerarin and ectoine.

[0011] Preferably, the active substance is a mixture of rhamnosyl uridine, silymarin, puerarin and ectoine in a molar ratio of (0.5-3): (0.2-2): (0.2-2.5): (0.05-1.5).

[0012] The inventors of the present application have discovered that adding active ingredients can further enhance the antioxidant and immunomodulatory effects of the collagen composite hemostatic sponge to a certain extent, reduce inflammation, oxidative stress, and cell damage, thereby increasing the wound healing rate and further enhancing biological activity.

[0013] The inventors of the present application have found that, in some cases, the use of the above-mentioned preferred scaffold proteins and polypeptides, supplemented with active substances as additives, can further improve the various performance indicators of the collagen composite hemostatic sponge, thereby enabling it to have more outstanding hemostatic ability, liquid absorption capacity and biocompatibility and other properties.

[0014] In a second aspect, the present application also provides a method for preparing the above-mentioned collagen composite hemostatic sponge, comprising the following steps: mixing a polypeptide solution, a collagen solution and a scaffold protein solution, incubating, then eliminating bubbles, performing self-assembly (which can be transferred to a mold), then removing excess water, precooling, and then freeze-drying.

[0015] In some preferred implementations, the preparation method of the above-mentioned collagen composite hemostatic sponge includes route one or route two;

[0016] The process of route 1 includes: mixing a solution of a peptide, a solution of a collagen protein, and a solution of a scaffold protein, incubating, then eliminating bubbles, performing self-assembly, adding a solution of an active substance, then removing excess water, precooling, and then freeze-drying;

[0017] The process of route 1 includes: mixing a solution of a peptide, a solution of a collagen protein and a solution of a scaffold protein, incubating, then eliminating bubbles, self-assembling, then removing excess water, precooling, then freeze-drying, adding a solution of an active substance, and then drying;

[0018] The active substances in line 1 and line 2 include at least one of rhamnosyl uridine, silymarin, puerarin and ectoine.

[0019] In some preferred implementations, the solution of the active substance is at least one of a 1mmol / L-2mmol / L ectoine solution, a 1mmol / L-2mmol / L rhamnosyl uridine solution, a 1mmol / L-2mmol / L silymarin solution, and a 1mmol / L-2mmol / L puerarin solution.

[0020] Preferably, the solution of the active substance is 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and the volume ratio thereof is: (0.5-3): (0.2-2): (0.2-2.5): (0.05-1.5).

[0021] In some preferred implementations, the concentration of the collagen solution is 5 mg / mL-10 mg / mL; the concentration of the polypeptide solution is 5 mg / mL-8 mg / mL; the concentration of the scaffold protein solution is 2 mg / mL-5 mg / mL; the volume ratio of the collagen solution, the polypeptide solution and the scaffold protein solution is (1-5): (0.5-2): (0.25-2).

[0022] Preferably, the concentration of the collagen solution is 5 mg / mL-10 mg / mL; the concentration of the polypeptide solution is 5 mg / mL-8 mg / mL; the concentration of the scaffold protein solution is 2 mg / mL-5 mg / mL; the volume ratio of the collagen solution, the polypeptide solution and the scaffold protein solution is (1-5): (0.5-2): (0.5-2).

[0023] In some preferred implementations, the incubation time is 3 h-5 h, and the temperature is 30° C.-40° C.

[0024] In some preferred implementations, the precooling temperature is -30°C to -80°C, and the precooling time is 2h to 12h.

[0025] In some preferred implementations, the freeze-drying temperature is -50°C to -60°C, the time is 24h to 72h, and the vacuum degree is -0.1MPa;

[0026] In a third aspect, the present application also provides the use of the above-mentioned collagen composite hemostatic sponge in medical supplies.

[0027] The beneficial effects of the present application are as follows: the collagen composite hemostatic sponge provided by the present application avoids the residue of cross-linking agents in traditional technologies, can not only absorb a large amount of liquid, but also has a strong adhesion ability to platelets and blood cells, which is beneficial to the activation of platelets and the activation of blood cell coagulation process, thereby achieving rapid coagulation; at the same time, the collagen composite hemostatic sponge provided by the present application also has excellent biocompatibility, and is an absorbable hemostatic material, which avoids frequent replacement and reduces patient pain. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments to clearly and completely describe the concept and technical effects of this application, so as to fully understand the purpose, scheme and effect of this application. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0029] The collagen solution in the following content is prepared by the following process:

[0030] The pig skin is defatted and freeze-dried; the freeze-dried pig skin is hydrolyzed in pepsin, then mixed with octyl glucosinolate and CHAPS (3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt), and the filtrate is filtered to obtain the filtrate; the filtrate is mixed with sodium chloride and salted out until a flocculent precipitate appears; the precipitate is redissolved, and purified and dialyzed in acetic acid and deionized water in sequence to obtain a collagen solution.

[0031] Example 1

[0032] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0033] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0034] 8 mg / mL collagen solution, 6 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0035] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 1:1.5:1:0.5 to prepare the active substance solution.

[0036] At 4°C, 8 mg / mL collagen, 6 mg / mL polypeptide solution and 3 mg / mL scaffold protein solution were fully mixed in a volume ratio of 2:1:0.5, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 8 hours. Subsequently, the solution of the active substance was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0037] Example 2

[0038] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0039] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0040] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0041] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.2:0.05 to prepare the active substance solution.

[0042] At 4°C, 8 mg / mL collagen, 8 mg / mL polypeptide solution and 3 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the solution of the active substance was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0043] Example 3

[0044] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0045] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0046] A 5 mg / mL collagen solution, a 5 mg / mL polypeptide (Fibronectin Active Fragment Control) solution, and a 2 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0047] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 3:2:2.5:1.5 to prepare the active substance solution.

[0048] At 4°C, 5 mg / mL collagen solution, 5 mg / mL polypeptide solution and 2 mg / mL scaffold protein solution were fully mixed in a volume ratio of 5:2:2, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the solution was transferred to the mold and allowed to self-assemble for 12 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0049] Example 4

[0050] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0051] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0052] A 10 mg / mL collagen solution, an 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution, and a 5 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0053] Prepare 2 mmol / L rhamnosyluridine DMSO solution, 2 mmol / L silymarin DMSO solution, 2 mmol / L puerarin DMSO solution and 2 mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 1:1:1:1 to prepare the active substance solution.

[0054] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1:1.5:2, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 8 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0055] Example 5

[0056] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin and puerarin; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0057] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0058] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0059] 2 mmol / L rhamnosyluridine DMSO solution, 2 mmol / L silymarin DMSO solution and 2 mmol / L puerarin DMSO solution were prepared, and the above solutions were mixed in a volume ratio of 0.5:0.2:0.2 to serve as the active substance solution.

[0060] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0061] Example 6

[0062] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0063] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0064] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0065] 2 mmol / L rhamnosyluridine DMSO solution, 2 mmol / L puerarin DMSO solution and 2 mmol / L ectoine aqueous solution were prepared, and the above solutions were mixed in a volume ratio of 0.5:0.2:0.05 to serve as the active substance solution.

[0066] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 8 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0067] Example 7

[0068] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include silymarin, puerarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0069] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0070] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0071] Prepare 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.2:0.2:0.05 to prepare the active substance solution.

[0072] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0073] Example 8

[0074] A collagen composite hemostatic sponge, the raw materials of which include polypeptide, collagen, scaffold protein and active substances, wherein the active substances include rhamnosyl uridine, silymarin and ectoine; the polypeptide, collagen and scaffold protein are self-assembled and then composited with the active substances.

[0075] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0076] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0077] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.05 to serve as the active substance solution.

[0078] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1:5:0.5:12, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 8 hours. Subsequently, the solution of the active substance was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0079] Example 9

[0080] A collagen composite hemostatic sponge is obtained by self-assembly of raw materials including polypeptide, collagen and scaffold protein.

[0081] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0082] 8 mg / mL collagen solution, 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0083] At 4°C, 10 mg / mL collagen solution, 8 mg / mL polypeptide solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 8 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0084] Comparative Example 1

[0085] The utility model relates to a pure collagen hemostatic sponge, which is made from collagen.

[0086] The preparation method of the pure collagen hemostatic sponge comprises the following steps:

[0087] 8 mg / mL collagen solution was prepared, mixed with distilled water at a volume ratio of 5:2 at 4°C, and incubated in a 33°C constant temperature water bath for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0088] Comparative Example 2

[0089] A collagen composite hemostatic sponge is obtained by self-assembly of raw materials including polypeptides, collagen and scaffold proteins; and active substances including rhamnosyl uridine, silymarin, puerarin and ectoine are added.

[0090] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0091] 2 mg / mL collagen solution, 2 mg / mL polypeptide (Fibronectin Active Fragment Control) solution and 1 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0092] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.2:0.05 to prepare the active substance solution.

[0093] At 4°C, 2 mg / mL collagen solution, 2 mg / mL polypeptide solution and 1 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the solution was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the solution of the active substance was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0094] Comparative Example 3

[0095] A collagen composite hemostatic sponge is obtained by self-assembly of raw materials including polypeptides, collagen and scaffold proteins; and active substances including rhamnosyl uridine, silymarin, puerarin and ectoine are added.

[0096] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0097] A 15 mg / mL collagen solution, a 13 mg / mL polypeptide (Fibronectin Active Fragment Control) solution, and an 8 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0098] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.2:0.05 to prepare the active substance solution.

[0099] At 4°C, 15 mg / mL collagen solution, 13 mg / mL polypeptide solution and 8 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:0.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the mixture was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0100] Comparative Example 4

[0101] A collagen composite hemostatic sponge is obtained by self-assembly of raw materials including collagen and scaffold protein; and active substances including rhamnosyl uridine, silymarin, puerarin and ectoine are added.

[0102] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0103] 8 mg / mL collagen solution and 3 mg / mL scaffold protein (RACK1 protein) solution were prepared and stored at 4°C.

[0104] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.2:0.05 to prepare the active substance solution.

[0105] At 4°C, 10 mg / mL collagen solution and 5 mg / mL scaffold protein solution were fully mixed in a volume ratio of 1.5:1, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the solution was transferred to the mold and allowed to self-assemble for 12 hours. Subsequently, the solution of the active substance was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0106] Comparative Example 5

[0107] A collagen composite hemostatic sponge is obtained by self-assembly of raw materials including polypeptide and collagen; and active substances including rhamnosyl uridine, silymarin, puerarin and ectoine are added.

[0108] The preparation method of the above-mentioned collagen composite hemostatic sponge comprises the following steps:

[0109] 8 mg / mL collagen solution and 8 mg / mL polypeptide (Fibronectin Active Fragment Control) solution were prepared and stored at 4°C.

[0110] Prepare 2mmol / L rhamnosyluridine DMSO solution, 2mmol / L silymarin DMSO solution, 2mmol / L puerarin DMSO solution and 2mmol / L ectoine aqueous solution, and mix the above solutions in a volume ratio of 0.5:0.2:0.2:0.05 to prepare the active substance solution.

[0111] At 4°C, 10 mg / mL collagen solution and 8 mg / mL polypeptide solution were fully mixed in a volume ratio of 1.5:0.5, and the mixed solution was placed in a 33°C constant temperature water bath for incubation for 3 hours. After incubation, ultrasonic vibration and centrifugation were performed to remove bubbles, and then the solution was transferred to a mold and allowed to self-assemble for 12 hours. Subsequently, the active substance solution was added, and after the active substance was loaded, it was placed in distilled water for dialyzation for 24 hours, and then pre-cooled at -80°C for 12 hours, and finally freeze-dried at -60°C and -0.1MPa for 24 hours.

[0112] Water absorption test

[0113] Take each embodiment and comparative example as a test sample, accurately weigh the mass of each test sample, record it as m1, and then completely immerse it in deionized water for 2 hours to allow it to fully absorb water, take it out, and weigh its mass as m2 after it stops dripping. Calculate the water absorption rate according to the formula. The results are shown in Table 1.

[0114] Water absorption (%) = (m2-m1) / m1×100%

[0115] Table 1 Water absorption of composite protein sponge

[0116]

[0117]

[0118] According to Table 1, it can be found that each test sample exhibits excellent water absorption capacity. Compared with Comparative Example 1, Examples 1-4 have better water absorption capacity, indicating that the water absorption effect of the collagen composite hemostatic sponge provided by the present application is better than that of a single pure collagen sponge. Compared with Examples 5-9, the water absorption capacity of Example 2 is significantly improved, indicating that the active substance loaded in the collagen composite hemostatic sponge provided by the present application can effectively improve its water absorption capacity.

[0119] In vitro hemolysis test

[0120] Take each embodiment and comparative example as a test sample, accurately weigh 5 mg and put it into a test tube and place it in a constant temperature water bath at 37°C for 5 minutes. Add 200 μL of fresh Balb / c mouse blood containing an anticoagulant (0.1 mol / L, sodium citrate) to a test tube, incubate in a water bath for 1 hour, centrifuge and take the supernatant, and use an enzyme marker to test the absorbance value at 545nm. Physiological saline and Triton X-100 are used as negative and positive controls, respectively. Calculate the hemolysis rate: hemolysis rate = (A sample-A negative) / (A positive-A negative) × 100%. Wherein A sample is the absorbance value of the experimental group, A negative is the absorbance value of the negative control group, and A positive is the absorbance value of the positive control group. The test results are shown in Table 2.

[0121] Table 2 Hemolysis rate / %

[0122]

[0123]

[0124] As can be seen from Table 2, by comparing the collagen composite hemostatic sponges prepared in Examples 1-4 of the present application, the collagen composite hemostatic sponges prepared in Examples 5-9, and the pure collagen sponge prepared in Comparative Example 1, it was found that the hemolysis rates of all groups were lower than 5%, indicating that the sponges have good blood compatibility. Among them, the hemolysis rates of Examples 1-4 are lower than 2%. The hemolysis rate of a hemostatic sponge refers to the degree of damage to red blood cells when the hemostatic sponge comes into contact with blood, that is, the proportion of hemoglobin released due to the rupture of red blood cells. The hemolysis rate is an important indicator for evaluating the biocompatibility of a hemostatic sponge, and is directly related to the safety and effectiveness of the hemostatic material. Therefore, the hemolysis rate of Examples 1-4 of the present application is lower than 2%, indicating that it has better biocompatibility and is more helpful for patient recovery.

[0125] In vitro clotting time analysis

[0126] Take each embodiment and comparative example as a test sample, weigh 5 mg and put it into the bottom of a test tube and preheat it to 37°C, then add 1 mL of recalcified whole blood (preparation method: add 100 μL of 0.2 mol / L CaCl2 solution to each 1 mL of anticoagulated whole blood), start timing immediately after adding, gently tilt the test tube every 3 to 5 seconds, observe the blood flow state, until the test tube is tilted 90° and the blood no longer flows, that is, coagulation, and record the coagulation time. The anticoagulated whole blood without sample is used as a blank control group, and each sample is measured 3 times to calculate the average value, and the data are shown in Table 3.

[0127] In vitro blood coagulation index (BCI) analysis

[0128] Take each embodiment and comparative example as a test sample, weigh 5 mg and put it in the bottom of the test tube, and take 5 mg of gauze as a negative control group sample and put it in the bottom of the test tube; then heat the test tube containing the test sample, the test tube containing the negative control group sample, the sodium citrate CaCl2 solution and the anticoagulated rabbit blood in a 37°C water bath for 10 minutes, and add an appropriate amount of sodium citrate CaCl2 solution (wherein sodium citrate: CaCl2=9:1) to the anticoagulated rabbit whole blood to recalcify it. Take 50 μL of recalcified whole blood and add it to the test tube containing the test sample and the test tube containing the negative control group sample. After all samples were incubated at 37°C for 10 minutes, 10 mL of deionized water was added to terminate the reaction, unreacted free red blood cells were removed, and the supernatant was collected to measure its absorbance at a wavelength of 540 nm. The blank group was the absorbance of 10 mL of deionized water added to the recalcified whole blood, and the in vitro coagulation index was calculated. Repeat 3 times and take the average value. The data are shown in Table 3.

[0129] The formula for calculating the coagulation index BCI (%) is: (where: ODS refers to the absorbance of the experimental group, ODB refers to the absorbance of the blank group)

[0130] Table 3 In vitro coagulation time and coagulation index results

[0131] Group In vitro coagulation time / s In vitro coagulation index / % Example 1 381 23.46％ Example 2 323 22.92％ Example 3 406 25.02％ Example 4 395 24.81％ Example 5 593 35.49％ Example 6 548 38.87％ Example 7 549 42.67％ Example 8 582 37.97％ Example 9 684 46.87％ Comparative Example 1 804 50.59％ Comparative Example 2 481 31.61％ Comparative Example 3 439 30.18％ Comparative Example 4 503 34.21％ Comparative Example 5 537 36.47％ Negative control 934 73.64％ Blank control / 100％

[0132] It is not difficult to see from Table 3 that the composite collagen sponges prepared by Examples 1-4 show faster coagulation ability and shorter coagulation time. Compared with the negative control group (gauze), the composite collagen sponge samples prepared by Examples 1-4 all have good coagulation performance; Example 2 is compared with Examples 5-9, and Example 2 shows faster coagulation time and coagulation performance, indicating that the loaded active substance will affect the coagulation time and coagulation performance of the composite collagen sponge to varying degrees, and the loaded active substance is more conducive to the in vitro coagulation time and coagulation performance of the composite collagen sponge, indicating that the collagen composite hemostatic sponge provided by the present application has excellent procoagulant performance and can effectively stop bleeding.

[0133] In vitro cytotoxicity assay

[0134] Each embodiment and comparative example was taken as a test sample, and the in vitro cytotoxicity of the composite collagen sponge was studied by MTT method, and mouse fibroblast L929 was used for testing. The specific experimental process is as follows:

[0135] After each test sample was sterilized by ultraviolet irradiation for 2 hours, it was mixed with MEM high-glucose medium containing 10% fetal bovine serum, incubated at 37°C for 72 hours, and the supernatant was collected. After L929 cells were cultured in a 96-well cell culture plate with complete medium, the complete medium was replaced with the sample extract, and cultured at 37°C in a 5% carbon dioxide cell culture incubator for 24 hours, with 6 parallel samples in each group. After 24 hours of culture, the cell morphology was observed under a microscope, and then the sample extract was replaced with 50 μL MTT (1 mg / mL) solution per well. After culturing at 37°C in a 5% carbon dioxide cell culture incubator for 2 hours, the MTT solution was discarded, and then 100 μL of isopropanol solution was added to each well, and the plate was placed flat in an ELISA reader, shaken at medium speed for 30 seconds, and then the absorbance at 570 nm was read. At the same time, a control sample was set, the positive control sample was a complete medium containing 5% DMSO; the negative control sample was a complete medium with the same conditions as the test sample. The relative cell activity was calculated as follows:

[0136] After L929 cells were incubated in the extracts of each material group for 24 h, the cell activity was detected by MTT, and the relative activity of cells in each material experimental group after 24 h of culture was calculated by absorbance, and the data are recorded in Table 4.

[0137] Table 4 MTT method to determine the cell activity of composite collagen sponge

[0138] Group Cell activity / % Example 1 99.34±4.36 Example 2 99.61±3.42 Example 3 98.98±3.48 Example 4 99.24±2.62 Example 5 94.61±8.47 Example 6 92.31±2.43 Example 7 93.19±4.91 Example 8 95.67±9.36 Example 9 90.67±10.67 Comparative Example 1 82.36±5.62 Comparative Example 2 84.96±11.34％ Comparative Example 3 85.37±10.59％ Comparative Example 4 88.59±9.23％ Comparative Example 5 86.497±10.57％ Positive Control 20.67±3.12 Negative control 100％

[0139] As can be seen from Table 4, there is no abnormal change in the cell morphology of each embodiment and comparative example. None of them showed obvious cytotoxicity, and since they did not show obvious cytotoxicity, they were classified as level 1 cytotoxicity; at the same time, the cell activity of each embodiment provided in the present application was higher than 98%, among which the cell activity of embodiment 1, embodiment 2, and embodiment 4 was higher than 99%, which was extremely close to the negative control, indicating that the collagen composite hemostatic sponge provided in the present application would not hinder the growth of cells and had excellent biocompatibility.

[0140] Functional analysis of liver wound surface

[0141] Each embodiment and comparative example was used as a test sample, and SD rats were used for the experiment. After the rats were anesthetized by 2% isoflurane inhalation, the abdomen was prepared and disinfected, and the abdomen was opened. A 1 cm 2 The materials of each group were attached to the wounds of different sizes to stop bleeding, and the bleeding time and amount of each group were recorded.

[0142] Table 5 Bleeding time and bleeding volume of SD rats

[0143] Group In vivo hemostasis time / s Bleeding volume / g Example 1 166.27±22.57 0.43±0.21 Example 2 145.83±16.28 0.39±0.19 Example 3 156.84±18.83 0.42±0.17 Example 4 172.5±20.17 0.38±0.16 Example 5 232.51±18.69 0.62±0.13 Example 6 212.56±39.69 0.56±0.16 Example 7 268.17±37.68 0.63±0.12 Example 8 226.37±29.27 0.65±0.18 Example 9 256.57±32.69 0.68±0.21 Comparative Example 1 319.28±55.99 0.71±0.23 Comparative Example 2 346.64±45.38 0.73±0.12 Comparative Example 3 352.41±52.16 0.72±0.22 Comparative Example 4 306.57±24.61 0.69±0.25 Comparative Example 5 312.64±35.42 0.70±0.15

[0144] It is not difficult to see from Table 5 that in the rat liver wound bleeding model, the hemostatic effect of the samples in the embodiments of the present application is significantly better than that of the comparative samples, indicating that the collagen composite hemostatic sponge prepared in the embodiments of the present application has the performance of rapid and effective hemostasis. According to analysis, this is because the collagen composite hemostatic sponge provided by the present application has a uniform and fine sponge structure, thus significantly improving the hemostatic performance in the case of massive bleeding in patients simulated by the rat liver wound bleeding model.

[0145] Although the description of the present application has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims by reference to the attached claims in view of the prior art, thereby effectively covering the intended scope of the present application. In addition, the above describes the present application with the embodiments foreseeable by the inventor, the purpose of which is to provide a useful description, and those non-substantial changes to the present application that have not yet been foreseen may still represent equivalent changes to the present application.

Claims

1. A collagen composite hemostatic sponge, characterized in that: The invention comprises a first material, wherein the first material is self-assembled from raw materials comprising a polypeptide, collagen and a scaffold protein, and the mass ratio of the collagen, the polypeptide and the scaffold protein is (5-50): (2.5-16): (0.5-10).

2. The collagen composite hemostatic sponge according to claim 1, characterized in that: The invention also comprises an active substance; the active substance comprises at least one of rhamnosyl uridine, silymarin, puerarin and ectoine.

3. A method for preparing the collagen composite hemostatic sponge as claimed in claim 1, characterized in that: The preparation method comprises the following steps: mixing the polypeptide solution, the collagen solution and the scaffold protein solution, incubating, then eliminating bubbles, performing self-assembly, then removing excess water, precooling, and then freeze-drying.

4. The method for preparing a collagen composite hemostatic sponge according to claim 3, characterized in that: The preparation method includes route 1 or route 2; The process of route 1 comprises: mixing the solution of the polypeptide, the solution of the collagen and the solution of the scaffold protein, incubating, then eliminating bubbles, self-assembling, adding the solution of the active substance, then removing excess water, precooling, and then freeze-drying; The process of route 1 comprises: mixing the solution of the polypeptide, the solution of the collagen and the solution of the scaffold protein, incubating, then eliminating bubbles, self-assembling, then removing excess water, precooling, then freeze-drying, adding the solution of the active substance, and then drying; The active substance includes at least one of rhamnosyl uridine, silymarin, puerarin and ectoine.

5. The method for preparing a collagen composite hemostatic sponge according to claim 4, characterized in that: The solution of the active substance is at least one of a 1mmol / L-2mmol / L ectoine solution, a 1mmol / L-2mmol / L rhamnosyl uridine solution, a 1mmol / L-2mmol / L silymarin solution, and a 1mmol / L-2mmol / L puerarin solution.

6. The method for preparing a collagen composite hemostatic sponge according to claim 3 or 4, characterized in that: The concentration of the collagen solution is 5mg / mL-10mg / mL; the concentration of the polypeptide solution is 5mg / mL-8mg / mL; the concentration of the scaffold protein solution is 2mg / mL-5mg / mL; the volume ratio of the collagen solution, the polypeptide solution and the scaffold protein solution is (1-5): (0.5-2): (0.25-2).

7. The method for preparing a collagen composite hemostatic sponge according to claim 3 or 4, characterized in that: The incubation time is 3h-5h, and the temperature is 30°C-40°C.

8. The method for preparing a collagen composite hemostatic sponge according to claim 3 or 4, characterized in that: The precooling temperature is -30°C to -80°C, and the precooling time is 2h to 12h.

9. The method for preparing a collagen composite hemostatic sponge according to claim 3 or 4, characterized in that: The freeze drying temperature is -50°C to -60°C, the time is 24h to 72h, and the vacuum degree is -0.1MPa.

10. Use of the collagen composite hemostatic sponge according to any one of claims 1 to 2 or the collagen composite hemostatic sponge prepared by the preparation method according to any one of claims 3 to 9 in medical supplies.

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