Medical fibroin sponge material as well as preparation method and application thereof

Abstract

The invention provides a medical fibroin sponge material and a preparation method and application thereof, the medical fibroin sponge material is composed of an aerogel support and chitosan / polysulfobetaine metacrylic acid ester composite microspheres, the aerogel support is composed of micro-nano fibroin fibers, the chitosan / polysulfobetaine metacrylic acid ester composite microspheres are loaded on the support, and the composite microspheres are distributed on the aerogel support in a gradient mode. The preparation method comprises the following steps: dissolving fibroin by using a ternary solution, directly shearing at a high speed, cooperating with high-speed shearing, formic acid swelling and steam explosion to obtain fibroin micro-nano fibers, freeze-drying to obtain a sponge bracket, retaining part of a fibroin beta-folded structure by high-speed shearing and steam explosion to form a multi-stage network tightly entangled among the fibers, and preparing the fibroin micro-nano fibers into the sponge bracket. Platelet and red blood cells can be more efficiently and mechanically captured, and blood coagulation is accelerated.

Description

Technical Field

[0001] This invention relates to the field of medical polymer materials, specifically to a medical silk fibroin sponge material, its preparation method, and its application. Background Technology

[0002] Most traumas are accompanied by acute bleeding, and hemostasis is crucial for relieving pain and reducing mortality in such incidents. In recent years, traditional hemostatic materials, including inorganic, protein-based, polysaccharide-based, and synthetic materials, have been widely used in clinical practice.

[0003] Blood coagulation involves two steps. First, vasoconstriction causes platelets to adhere and aggregate, forming a hemostatic plug, achieving initial hemostasis. Then, a coagulation cascade initiates plasma coagulation, and the fibrin network strengthens the platelet thrombus, achieving the second stage of effective hemostasis. Therefore, successful hemostasis hinges on two key components: the coagulation cascade and platelet activation. In the coagulation cascade, the activation of coagulation factor X affects thrombin production, directly determining blood coagulation. Platelet activation influences hemostatic plug formation and also activates prothrombin to achieve blood coagulation.

[0004] To date, a wide range of hemostatic materials have emerged on the market. For example, α-cyanoacrylate and gelatin stop bleeding by physically sealing wounds or compressing blood vessels. There are also hemostatic biomaterials that form nano- or micro-sized pores, such as zeolite and mesoporous silica, as well as biopolymers like gelatin and starch. These concentrate certain components of the blood through physical or chemical action, accelerating the activation of clotting factors and thus stopping bleeding. Some hemostatic biomaterials can directly activate clotting factors or platelets to initiate the coagulation cascade reaction, such as chitosan, alginate, oxidized cellulose, and fibrin glue. Chitosan, when attached to a wound, can activate platelets and the complement system in the blood. Alginate, after reacting with sodium ions, can activate clotting factors. Oxidized cellulose, after rapidly dissolving in the blood, can activate various clotting factors and aggregate platelets, while fibrin glue can rapidly initiate the intrinsic coagulation system.

[0005] Silk fibroin is a natural high-molecular-weight fibrous protein. It contains a high content of amino acids with hydrophilic amino and carboxyl groups, and the hydrophilic groups in its non-crystalline regions contribute to its good air and water permeability and absorbency. Silk fibroin exhibits good biocompatibility, is non-irritating, non-toxic, has low inflammatory reactivity, and excellent chemical and physical properties, and possesses a certain degree of biodegradability. Silk fibroin has been used in artificial blood vessels, cell culture media, nerve tissue, ligaments, bone-compatible materials, and artificial skin. Research on the application of silk fibroin in hemostatic materials has also attracted considerable attention. Current research involves preparing porous silk fibroin materials by mixing silk fibroin with the cross-linking agent polyethylene glycol diglycidyl ether in a specific ratio and then cold-drying. However, the absorbency of the hemostatic material prepared in this way is far lower than that of commercially available gelatin sponges, resulting in low hemostatic efficiency and limited functionality, requiring further improvement. Summary of the Invention

[0006] Technical problem to be solved: The purpose of this invention is to provide a medical silk fibroin sponge material.

[0007] Technical solution: A medical silk fibroin sponge material, wherein the medical silk fibroin sponge material is composed of an aerogel scaffold made of micro and nano silk fibroin fibers and chitosan / polysulfobetaine methacrylate composite microspheres loaded on the scaffold, and the composite microspheres are gradient distributed on the aerogel scaffold.

[0008] The preparation method of the above-mentioned medical silk fibroin sponge material includes the following steps: S1. After degumming silk, silk fibroin fibers are obtained. The silk fibroin fibers are chopped and added to deionized water. The silk fibroin dispersion is obtained by high-speed shearing, and then filtered and dried to obtain micro-nano silk fibroin fibers. S2. The micro / nano silk fibers prepared in step S1 are added to formic acid solution for swelling. After swelling is complete, the solution is filtered to obtain the swollen micro / nano silk fibers. S3. The swollen micro / nano silk fibroin fibers prepared in step S2 are subjected to steam explosion, the micro / nano fibers after steam explosion are collected, washed and dried to obtain explosion-modified micro / nano silk fibroin fibers. S4. Add the burst-modified micro / nano silk fiber obtained in step S3 to ice water and stir to disperse evenly; S5. Place the micro / nano silk fiber dispersion obtained by stirring in step S4 under ice bath conditions, then add chitosan / polysulfobetaine methacrylate composite microspheres to the dispersion, stir and centrifuge, then freeze the centrifuged solution in liquid nitrogen and freeze-dry to obtain medical silk sponge material.

[0009] Preferably, the preparation method of the chitosan / polysulfobetaine methacrylate composite microspheres includes the following steps: S11. Dissolve low molecular weight water-soluble chitosan in warm water to obtain a chitosan solution with a concentration of 5~10wt%; S12. Dissolve polysulfobetaine methacrylate in warm water to obtain a polysulfobetaine methacrylate solution with a concentration of 5~10wt%; S13. Mix the two solutions to make the mass ratio of chitosan to polysulfobetaine methacrylate in the mixed solution 2~3:1. After the solution is stirred evenly, spray and dry to obtain chitosan / polysulfobetaine methacrylate composite microspheres.

[0010] Preferably, in step S1, the mass-to-volume ratio of the chopped silk fibroin fibers to deionized water is 30-80 g: 1000 mL; and / or, The high-speed shearing speed is 10000~15000 r / min, and the high-speed shearing time is 15~30 min.

[0011] Preferably, the concentration of the formic acid solution in step S2 is 65-80 wt%, and the swelling time is 15-40 min.

[0012] Preferably, the steam explosion pressure in step S3 is 1.2~1.5MPa, and the pressure holding time is 60~90s.

[0013] Preferably, in step S4, the concentration of the micro / nano silk fiber dispersion is 7.5~15.8 wt%, and the content of the chitosan / polysulfobetaine methacrylate composite microspheres in the dispersion is 1.5~3.2 wt%; and / or, The centrifugation speed is 800~1500 r / min, and the time is 2~5 min; and / or, The freeze-drying temperature is -60~-20℃, and the time is 15~40h.

[0014] Preferably, the low molecular weight water-soluble chitosan has a molecular weight of 1.5~2.2kDa, and the polysulfobetaine methacrylate has a molecular weight of 20~40kDa.

[0015] The above-mentioned medical silk fibroin sponge material is used in the preparation of hemostatic materials.

[0016] Beneficial effects: The medical silk fibroin sponge material of this invention has the following advantages: In the past, silk fibroin sponges were prepared by dialysis of silk fibroin protein solution using a ternary solution and freeze-drying. In the above method, the ternary solution is not used for dissolution. Instead, high-speed shearing is used directly, combined with high-speed shearing, formic acid swelling and steam explosion to obtain silk fibroin micro and nanofibers. Then, freeze-drying is used to obtain a sponge scaffold. High-speed shearing and steam explosion retain some of the β-sheet structure of silk fibroin, forming a multi-level network of tightly entangled fibers, which can more efficiently capture platelets and red blood cells and accelerate coagulation. The silk sponge material prepared by the present invention using micro-nano silk fiber scaffolds and chitosan / polysulfonated betaine methacrylate composite microspheres has a good overall hemostatic effect and is conducive to rapid hemostasis. The sponge material of this invention achieves microsphere loading through physical adsorption and freeze fixation, eliminating the need for toxic crosslinking agents such as glutaraldehyde, thus ensuring the biosafety of the material. Attached Figure Description

[0017] Figure 1 The appearance of hemostatic materials; Figure 2 Examples 1-4, Comparative Examples 1-2, Comparative Example 11, and commercially available gelatin sponges were compared at different times for water absorption rates. Figure 3 The water absorption rates at different times for Examples 1, 5-7 and Comparative Example 3; Figure 4 The water absorption rates at different times for Examples 1, 8-10 and Comparative Example 5; Figure 5 The water absorption rates at different times are for Examples 1, 11-13 and Comparative Example 6; Figure 6 The water absorption rates at different times are for Examples 1, 14-15 and Comparative Example 7; Figure 7 Platelet adhesion rates of Example 1, Comparative Examples 6-11, and commercially available gelatin sponges; Figure 8 The blood absorption time is measured for Example 1, Comparative Examples 6-11, and commercially available gelatin sponges. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: This invention provides a medical silk fibroin sponge material, comprising an aerogel scaffold composed of micro / nano silk fibroin fibers and chitosan / polysulfobetaine methacrylate composite microspheres loaded on the scaffold. The composite microspheres are gradient-distributed on the aerogel scaffold, with more microspheres aggregated at the skin-contact surface. The aerogel scaffold, constructed from micro / nano silk fibroin fibers, forms a three-dimensional network through physical entanglement and hydrogen bonding, resulting in superior mechanical properties compared to traditional silk fibroin sponges. Furthermore, the aerogel prepared from micro / nano fibers contains both macropores and micropores. Macropores can rapidly absorb blood and concentrate clotting factors, while micropores can absorb blood through capillary action. This pore structure is superior to the single pore size of homogeneous sponges. The composite microspheres, prepared from chitosan and polysulfobetaine methacrylate, are more abundant at the skin-contact surface. Chitosan releases protonated amino groups (-NH3) on the wound-contact side. + Polysulfobetaine methacrylate (PSMA) adsorbs negatively charged red blood cells and activates platelets through electrostatic interactions, while providing antibacterial protection. This endows the microspheres with excellent anti-protein adsorption and antibacterial adhesion properties, reducing the risk of infection.

[0019] The preparation method of the above-mentioned medical silk fibroin sponge material includes the following steps: S1. After degumming silk, silk fibroin fibers are obtained. The silk fibroin fibers are chopped and added to deionized water. The silk fibroin dispersion is obtained by high-speed shearing, and then filtered and dried to obtain micro-nano silk fibroin fibers. S2. The micro / nano silk fibers prepared in step S1 are added to formic acid solution for swelling. After swelling is complete, the solution is filtered to obtain the swollen micro / nano silk fibers. S3. The swollen micro / nano silk fibroin fibers prepared in step S2 are subjected to steam explosion, the micro / nano fibers after steam explosion are collected, washed and dried to obtain explosion-modified micro / nano silk fibroin fibers. S4. Add the burst-modified micro / nano silk fiber obtained in step S3 to ice water and stir to disperse evenly; S5. Place the micro / nano silk fiber dispersion obtained by stirring in step S4 under ice bath conditions, then add chitosan / polysulfobetaine methacrylate composite microspheres to the dispersion, stir and centrifuge, then freeze the centrifuged solution in liquid nitrogen and freeze-dry to obtain medical silk sponge material.

[0020] In step S1 above, the silk fibroin fibers are pulverized into micro-nano fibers through high-speed shearing. Moreover, high-speed shearing avoids the chemical modification of silk fibroin proteins and does not require any chemical reagents, thus obtaining micro-nano fibers simply and efficiently. In step S2, formic acid is used to swell the silk fibroin. Formic acid is an organic acid solvent that can penetrate the amorphous region of the silk fibroin fiber, destroy the β-sheet hydrogen bond network, swell the fiber and expose more active sites, providing structural defects for subsequent steam explosion. At the same time, after swelling, the hydroxyl (-OH) and amino (-NH2) groups on the fiber surface are exposed, which enhances the physical adsorption or chemical bonding ability between fibers and between fibers and composite microspheres. In step S3, the micro- and nano-fibers treated with formic acid are directly released with high-pressure steam without washing, causing the moisture inside the fiber to vaporize and expand, bursting from the inside out, further dissociating the micro-fibers into nanofibers, increasing the specific surface area and porosity of the scaffold, and improving the adsorption performance of the sponge material. Steps S4 and S5 are both carried out under ice bath conditions. This is because polysulfobetaine methacrylate is easily soluble in aqueous solutions at temperatures above 20°C, but will not dissolve in an ice bath. Centrifugation is used to cause the denser composite microspheres to settle towards the contact surface, forming a gradient distribution of "sparse on top and dense on the bottom", which directly enhances the antibacterial, lubricating and drug release functions of the contact surface. Liquid nitrogen quick-freezing can quickly lock this gradient distribution structure.

[0021] Preferably, the preparation method of the chitosan / polysulfobetaine methacrylate composite microspheres includes the following steps: S11. Dissolve low molecular weight water-soluble chitosan in warm water to obtain a chitosan solution with a concentration of 5~10wt%; S12. Dissolve polysulfobetaine methacrylate in warm water to obtain a polysulfobetaine methacrylate solution with a concentration of 5~10wt%; S13. Mix the two solutions to make the mass ratio of chitosan to polysulfobetaine methacrylate in the mixed solution 2~3:1. After the solution is stirred evenly, spray and dry to obtain chitosan / polysulfobetaine methacrylate composite microspheres.

[0022] The preparation of the above-mentioned composite microspheres does not use traditional cross-linking agents (such as glutaraldehyde and genipin) which may leave toxic groups (such as aldehyde groups). The physical mixing method completely avoids the introduction of chemical cross-linking agents, ensuring the biosafety of the material when it comes into contact with the wound.

[0023] Chitosan and polysulfonated betaine methacrylate form a complex through electrostatic attraction. During spray drying, rapid evaporation of moisture causes the two to become tightly intertwined, forming a stable microsphere structure. The amino groups (-NH2) of chitosan and the sulfonic acid groups (-SO3) of polysulfonated betaine methacrylate... - It was not chemically modified due to the cross-linking reaction, thus retaining the natural functional groups of both, ensuring the full utilization of chitosan's antibacterial and anti-adhesion properties.

[0024] In one embodiment, the mass-to-volume ratio of the chopped silk fibroin fibers to deionized water in step S1 is 30-80 g: 1000 mL. Maintaining a suitable mass-to-volume ratio between the silk fibroin fibers and the solvent is beneficial to improving the uniformity of the micro- and nano-silk fibroin fibers obtained during high-speed shearing. The high-speed shearing speed is 10,000~15,000 r / min, and the high-speed shearing time is 15~30 min. The high-speed shearing dissociates the short-cut millimeter-sized fibers into micro-nano-sized fibers through a mechanical force field, which greatly increases the specific surface area and enhances the efficiency of subsequent swelling and bursting.

[0025] In one embodiment, the concentration of the formic acid solution in step S2 is 65-80 wt%, and the swelling time is 15-40 min. Formic acid has low solubility for silk fibroin. Using formic acid with a concentration range of 65-80 wt% will cause the formic acid to swell the micro and nano silk fibroin fibers. The acid in this range can both prevent excessive hydrolysis of the fibers and maintain their mechanical integrity, and can also destroy some of the β-sheet hydrogen bond network, causing the fibers to swell and expose more active sites.

[0026] In one embodiment, the steam explosion pressure in step S3 is 1.2~1.5MPa, and the pressure holding time is 60~90s. The appropriate explosion pressure quickly penetrates the amorphous region of the silk fiber, weakens hydrogen bonds and hydrophobic interactions, and avoids local over-dissociation or incomplete dissociation.

[0027] The centrifugation speed is 800~1500 r / min and the time is 2~5 min. Appropriate centrifugal force causes the composite microspheres with higher density to settle towards the contact surface, forming a gradient distribution of "sparse on top and dense on the bottom". However, if the centrifugal force is too large or too small, the composite microspheres will be too dense on the contact surface or will not achieve the gradient distribution effect. In one embodiment, the low molecular weight water-soluble chitosan has a molecular weight of 1.5~2.2kDa, and the polysulfobetaine methacrylate has a molecular weight of 20~40kDa. The low molecular weight water-soluble chitosan can rapidly absorb water and swell, forming an antibacterial protective layer on the bleeding surface. When the polysulfobetaine methacrylate comes into contact with the bleeding wound, the body temperature and the temperature of the blood will cause the polysulfobetaine methacrylate to form a surface hydration layer through strong hydration after absorbing the blood. This can maintain a moist environment on the wound surface, promote autolytic debridement, and its anti-protein adsorption properties can reduce bacterial adhesion. Example

[0028] The preparation method of medical silk fibroin sponge material includes the following steps: S1. After degumming silk, silk fibroin fibers are obtained. The silk fibroin fibers are chopped and added to deionized water at a mass-to-volume ratio of 30g:1000mL. The mixture is then subjected to high-speed shearing at a speed of 12000r / min for 20min to obtain a silk fibroin dispersion. The dispersion is then filtered and dried to obtain micro / nano silk fibroin fibers. S2. Add the micro / nano silk fibers prepared in step S1 to a 70wt% formic acid solution to swell for 25 min. After swelling is complete, filter to obtain the swollen micro / nano silk fibers. S3. The swollen micro / nano silk fibers prepared in step S2 are subjected to steam explosion at a pressure of 1.2 MPa and a holding time of 90 s. The micro / nano fibers after steam explosion are collected, washed and dried to obtain explosion-modified micro / nano silk fibers. S4. Add the burst-modified micro / nano silk fiber obtained in step S3 to ice water and stir to disperse evenly; S5. Place the 10.0wt% micro / nano silk fiber dispersion obtained in step S4 under ice bath conditions, and then add chitosan / polysulfonated betaine methacrylate composite microspheres to the dispersion. The content of chitosan / polysulfonated betaine methacrylate composite microspheres in the dispersion is 2.1wt%. After stirring, centrifuge at 1100 r / min for 4 min. Quickly freeze the centrifuged solution in liquid nitrogen and then freeze-dry it at -40℃ for 25 h to obtain medical silk fibroin sponge material.

[0029] Preferably, the preparation method of the chitosan / polysulfobetaine methacrylate composite microspheres includes the following steps: S11. Select water-soluble chitosan with a molecular weight of 1.5~2.2kDa and dissolve it in warm water to obtain a chitosan solution with a concentration of 7.5wt%. S12. Polysulfobetaine methacrylate with a molecular weight of 20~30kDa was dissolved in warm water to obtain a polysulfobetaine methacrylate solution with a concentration of 7.5wt%. S13. Mix the two solutions to make the mass ratio of chitosan to polysulfobetaine methacrylate in the mixed solution 2:1. After the solution is stirred evenly, spray and dry to obtain chitosan / polysulfobetaine methacrylate composite microspheres. Example

[0030] The difference between Example 2 and Example 1 is that the rotation speed of the high-speed shearing in step S1 is 10000 r / min, and the high-speed shearing time is 15 min. Example

[0031] The difference between Example 3 and Example 1 is that the rotation speed of the high-speed shearing in step S1 is 14000 r / min, and the high-speed shearing time is 25 min. Example

[0032] The difference between Example 4 and Example 1 is that the rotation speed of the high-speed shearing in step S1 is 15000 r / min, and the high-speed shearing time is 30 min. Example

[0033] The difference between Example 5 and Example 1 is that the concentration of formic acid in step S2 is 65 wt%, and the swelling time is 15 min. Example

[0034] The difference between Example 6 and Example 1 is that the concentration of formic acid in step S2 is 75 wt%, and the swelling time is 30 min. Example

[0035] The difference between Example 7 and Example 1 is that the concentration of formic acid in step S2 is 80 wt%, and the swelling time is 40 min. Example

[0036] The difference between Example 8 and Example 1 is that the steam explosion pressure in step S3 is 1.3 MPa and the pressure holding time is 90 s. Example

[0037] The difference between Example 9 and Example 1 is that the steam explosion pressure in step S3 is 1.4 MPa and the pressure holding time is 90 s. Example

[0038] The difference between Example 10 and Example 1 is that the steam explosion pressure in step S3 is 1.5 MPa and the pressure holding time is 90 s. Example

[0039] The difference between Example 11 and Example 1 is that the concentration of the micro / nano silk fiber dispersion is 7.5 wt%, and the content of chitosan / polysulfobetaine methacrylate composite microspheres in the dispersion is 1.5 wt%. Example

[0040] The difference between Example 12 and Example 1 is that the concentration of the micro / nano silk fiber dispersion is 15.8 wt%, and the content of chitosan / polysulfonated betaine methacrylate composite microspheres in the dispersion is 3.2 wt%. Example

[0041] The difference between Example 13 and Example 1 is that the concentration of the micro / nano silk fiber dispersion is 15.8 wt%, and the content of chitosan / polysulfonated betaine methacrylate composite microspheres in the dispersion is 3.2 wt%. Example

[0042] The difference between Example 14 and Example 1 is that the centrifugation speed is 800 r / min. Example

[0043] The difference between Example 15 and Example 1 is that the centrifugation speed is 1500 r / min.

[0044] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the rotation speed of the high-speed shearing in step S1 is 20000 r / min, and the high-speed shearing time is 30 min.

[0045] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the rotation speed of the high-speed shearing in step S1 is 8000 r / min, and the high-speed shearing time is 10 min.

[0046] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the concentration of formic acid in step S2 is 98 wt%, and the swelling time is 15 min.

[0047] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the steam explosion pressure in step S3 is 1.0 MPa and the pressure holding time is 40 s.

[0048] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the steam explosion pressure in step S3 is 3.0 MPa and the pressure holding time is 90 s.

[0049] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that the concentration of the micro / nano silk fiber dispersion is 5 wt%, and the content of chitosan / polysulfobetaine methacrylate composite microspheres in the dispersion is 1.5 wt%.

[0050] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the centrifugation speed is 5000 r / min.

[0051] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that the medical silk fibroin sponge material contains only an aerogel scaffold composed of micro-nano silk fibroin fibers.

[0052] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that the molecular weight of the water-soluble chitosan is 10~15kDa.

[0053] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that chitosan is not added to the composite microspheres.

[0054] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that the aerogel scaffold is made by lyophilizing a silk fibroin solution dissolved in a ternary solution after dialysis and concentration to obtain a 10wt% silk fibroin protein solution.

[0055] Performance testing: Test Example 1 Water absorption: Take 3 parallel samples of each of the above-prepared materials, weigh m1 first, and then weigh m2 of the material after immersion in water for 10s, 30s and 60s respectively. Calculate the water absorption rate m of the material (repeat 3 times for each material): m (%) = (m2-m1) / m1*100%.

[0056] according to Figure 2 It can be seen that shear parameters have a significant impact on the water absorption performance of sponge materials. High-speed shearing can pulverize silk fibroin fibers into micro- and nano-sized silk fibroin fibers, and the water absorption performance increases with increasing shear rate. Furthermore, the short-term water absorption rate of micro- and nano-sized silk fibroin fibers is greater than that of porous silk fibroin materials and commercially available gelatin sponges. Figure 3 As can be seen, the concentration of formic acid also has a certain impact on the water absorption rate. High concentrations of formic acid will cause the micro / nano silk fibers to shrink, leading to a decrease in water absorption rate; from Figure 4 It can be seen that the higher the steam explosion pressure and the longer the time, the higher the water absorption rate. However, to ensure the mechanical properties of the sponge material, it is necessary to select appropriate explosion pressure and time. Figure 5 As can be seen, the appropriate concentration of micro / nano silk fibers and chitosan / polysulfobetaine methacrylate composite microspheres also affects the water absorption performance; from Figure 6 As can be seen, excessively high centrifugal speed causes the micro- and nano-silk fibers and composite microspheres to accumulate together, which is not conducive to water absorption.

[0057] Test Example 2 Pore ​​size distribution: The pore size distribution of the sponge material was tested using a NOVA4200e fully automatic high-speed specific surface area porosity analyzer.

[0058] Table 1 shows the pore size distribution in sponge materials. Large pores (>200um) Micropores (<100 nm) Example 1 56.5% 15.5% Comparative Example 7 46.2% 13.8% Comparative Example 11 38.9% 11.1% As can be seen from the table above, the sponge material obtained in Example 1 has a large number of macropores, which is conducive to the rapid water absorption of the sponge material and allows water molecules to quickly penetrate into the interior of the sponge material. Excessive centrifugation will cause the micro-nano fibers to aggregate, resulting in a decrease in the proportion of macropores. The proportion of micropores in Example 1 is also better than that in Comparative Example 7 and Comparative Example 11. A higher micropore content is conducive to capillary water absorption.

[0059] Test Example 3 Expansion rate: Take 3 parallel samples of each of the above-prepared materials, first measure the volume of the material V0, then let the material be completely immersed in water, drain it after soaking, put it in a certain volume of deionized water and record it as V1, and measure the amount of water rise in the container V2 (repeat 3 times for each material): V (%) = (V2-V1-V0) / V0*100%.

[0060] Table 2 shows the expansion rate of sponge materials. Expansion rate (%) Example 1 20.8% Comparative Example 8 18.3% Comparative Example 9 21.6% Comparative Example 10 19.1% Comparative Example 11 12.5% Commercially available gelatin sponges 39.6% As can be seen from Table 2, the expansion rate of the sponge material prepared in Example 1 is slightly lower than that of commercially available gelatin sponges, but the combination... Figure 2 As can be seen, the instantaneous water absorption effect of the sponge material prepared in Example 1 is better than that of commercially available gelatin sponge, and the expansion rate of the sponge material in Example 1 is better than that of the sponge material prepared directly with silk protein solution.

[0061] Test Example 4 Compression performance test: The compression performance test is conducted using a material testing machine. The upper pressure plate has a sensor, and the lower end is a platform for placing materials. The compressibility of the material is detected by measuring the force required to press down 6mm when the upper pressure plate comes into contact with the material. Each material is tested three times.

[0062] Table 3 shows the compressibility of sponge materials. Load capacity (N) Load capacity (N) Example 1 11.9 Comparative Example 1 11.5 Example 2 11.2 Comparative Example 2 10.9 Example 3 11.8 Comparative Example 3 12.5 Example 4 12.2 Comparative Example 4 10.9 Example 5 11.6 Comparative Example 5 10.4 Example 6 12.2 Comparative Example 6 10.6 Example 7 12.6 Comparative Example 7 12.6 Example 8 12.1 Comparative Example 8 11.8 Example 9 12.5 Comparative Example 9 12.1 Example 10 12.6 Comparative Example 10 12.3 Example 11 11.1 Comparative Example 11 10.8 Example 12 12.4 Commercially available gelatin sponges 3.2 Example 13 12.6 Example 14 12.1 Example 15 12.0 As can be seen from Table 4, shear rate, formic acid concentration, steam explosion pressure, and the content of micro / nano silk fibers all affect the compressibility of sponge materials.

[0063] Test Example 5 Platelet adhesion rate test: Fresh rabbit blood was centrifuged for 15 min at 1500 r / min, and platelet-rich plasma (PRP) was collected and counted. The PRP to be inoculated was diluted with PBS solution (5×10⁻⁶). 5Platelet adhesion rate (%). Add 1 ml of diluted PRP solution to each well of the material placed in the 48-well plate. Incubate for 3 hours, then remove and calculate the number of platelets in each well. Finally, calculate the adhesion rate using the adhesion rate formula. Perform three replicates for each sample. The amount of platelets added to each well is N1, and the number of platelets in the solution after adhesion is N2: Platelet adhesion rate (%) = (N1 - N2) / N1 * 100%.

[0064] from Figure 7 As can be seen from the results, the platelet adhesion rate of commercially available gelatin sponge material is the lowest, followed by the sponge material prepared by the method of Comparative Example 11. In Comparative Example 6, the platelet adhesion rate is relatively low due to the low content of composite microspheres in the solution. From Comparative Examples 7 to 10, it can be seen that the amount and distribution of chitosan content are the key factors affecting the platelet adhesion rate.

[0065] Test Example 6 Hemostasis test: Make a longitudinal incision in the skin of the rabbit's ear with a scalpel, quickly cover the wound with the material, and gently pinch it for a period of time until no more blood flows from the wound. Use a timer to determine the hemostasis time. The wound is 1 cm long. Record the hemostasis time and evaluate the effectiveness of hemostasis.

[0066] Depend on Figure 8 It can be seen that the hemostasis time of Example 1 is about 20 seconds. When the small-molecule chitosan comes into contact with blood, it can quickly absorb water and hydrolyze, forming a network gel on the wound surface, which accelerates blood coagulation and shortens bleeding time. In Comparative Example 9, due to the large molecular weight of chitosan, it cannot quickly absorb water and hydrolyze, resulting in a less effective hemostasis than that of Example 1.

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A medical-grade silk fibroin sponge material, characterized in that: The medical silk fibroin sponge material consists of an aerogel scaffold made of micro and nano silk fibroin fibers and chitosan / polysulfobetaine methacrylate composite microspheres loaded on the scaffold, with the composite microspheres distributed in a gradient on the aerogel scaffold.

2. The method for preparing the medical silk fibroin sponge material according to claim 1, characterized in that, Includes the following steps: S1. After degumming silk, silk fibroin fibers are obtained. The silk fibroin fibers are chopped and added to deionized water. The silk fibroin dispersion is obtained by high-speed shearing, and then filtered and dried to obtain micro-nano silk fibroin fibers. S2. The micro / nano silk fibers prepared in step S1 are added to formic acid solution for swelling. After swelling is complete, the solution is filtered to obtain the swollen micro / nano silk fibers. S3. The swollen micro / nano silk fibroin fibers prepared in step S2 are subjected to steam explosion, the micro / nano fibers after steam explosion are collected, washed and dried to obtain explosion-modified micro / nano silk fibroin fibers. S4. Add the burst-modified micro / nano silk fiber obtained in step S3 to ice water and stir to disperse evenly; S5. Place the micro / nano silk fiber dispersion obtained by stirring in step S4 under ice bath conditions, then add chitosan / polysulfobetaine methacrylate composite microspheres to the dispersion, stir and centrifuge, then freeze the centrifuged solution in liquid nitrogen and freeze-dry to obtain medical silk sponge material.

3. The method for preparing medical silk fibroin sponge material according to claim 2, characterized in that: The preparation method of the chitosan / polysulfonated betaine methacrylate composite microspheres includes the following steps: S11. Dissolve low molecular weight water-soluble chitosan in warm water to obtain a chitosan solution with a concentration of 5~10wt%; S12. Dissolve polysulfobetaine methacrylate in warm water to obtain a polysulfobetaine methacrylate solution with a concentration of 5~10wt%; S13. Mix the two solutions to make the mass ratio of chitosan to polysulfobetaine methacrylate in the mixed solution 2~3:

1. After the solution is stirred evenly, spray and dry to obtain chitosan / polysulfobetaine methacrylate composite microspheres.

4. The method for preparing medical silk fibroin sponge material according to claim 2, characterized in that: In step S1, the mass-to-volume ratio of the chopped silk fibroin fibers to deionized water is 30-80 g: 1000 mL; and / or, The high-speed shearing speed is 10000~15000 r / min, and the high-speed shearing time is 15~30 min.

5. The method for preparing medical silk fibroin sponge material according to claim 2, characterized in that: In step S2, the concentration of the formic acid solution is 65-80 wt%, and the swelling time is 15-40 min.

6. The method for preparing medical silk fibroin sponge material according to claim 2, characterized in that: In step S3, the steam explosion pressure is 1.2~1.5MPa, and the pressure holding time is 60~90s.

7. The method for preparing medical silk fibroin sponge material according to claim 2, characterized in that: In step S4, the concentration of the micro / nano silk fiber dispersion is 7.5~15.8 wt%, and the content of the chitosan / polysulfobetaine methacrylate composite microspheres in the dispersion is 1.5~3.2 wt%; and / or, The centrifugation speed is 800~1500 r / min, and the time is 2~5 min; and / or, The freeze-drying temperature is -60~-20℃, and the time is 15~40h.

8. The method for preparing medical silk fibroin sponge material according to claim 3, characterized in that: The low molecular weight water-soluble chitosan has a molecular weight of 1.5~2.2kDa, and the polysulfobetaine methacrylate has a molecular weight of 20~40kDa.

9. The application of the medical silk fibroin sponge material as described in claim 1 in the preparation of hemostatic materials.

Images