A multifunctional medical composite sponge and its preparation method and application

Abstract

The present invention relates to a multifunctional medical composite sponge and a preparation method and application thereof, belonging to the field of biomedical technology. The preparation method of the present invention comprises the following steps: adding a first silk fibroin solution and ethanol to a first mixed solution, washing and freeze-drying to obtain silk fibroin microspheres; the first mixed solution is obtained by dissolving calcium ascorbate and ascorbic acid in water; adding silk fibroin microspheres to a protamine solution, stirring, standing, centrifuging, and freeze-drying to obtain composite protein microspheres; after the second mixed solution is low-temperature balanced, a cross-linking agent and the composite protein microspheres are added to perform an oscillating cross-linking reaction, and freeze-drying to obtain a multifunctional medical composite sponge; the second mixed solution is obtained by mixing a second silk fibroin solution, a wool keratin solution, and a sodium hyaluronate solution. The sponge can induce a hemostatic cascade reaction while long-term liquid absorption and antibacterial and anti-inflammatory, achieving multiple hemostasis and having the potential to promote wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a multifunctional medical composite sponge and a preparation method and application thereof. Background Art

[0002] In daily life, trauma to the skin or tissue accompanied by bleeding is a common phenomenon that is difficult to completely avoid. When the trauma reaches a large range or depth, it may quickly lead to excessive blood loss in the human body, posing a serious threat to life. If the uncontrollable bleeding condition is not effectively controlled within the critical thirty minutes of emergency treatment, the risk of casualties will rise sharply. Therefore, the development of a new type of hemostatic material that can respond quickly and achieve hemostasis in multiple ways has become the core trend of the future development of hemostatic technology. The ideal hemostatic material should have the potential to quickly and efficiently control bleeding, long-term antibacterial ability, biodegradability, good biocompatibility and promote wound healing.

[0003] As a kind of widely used repair material, porous sponge plays an important role in the field of hemostasis treatment because of its large number of pore structures that are conducive to promoting body fluid absorption and tissue fluid exchange. Silk fibroin is a natural polymer protein extracted from silk, which has good biocompatibility and controllable biodegradability. Silk fibroin can be transformed into various forms of materials such as nanoparticles, films, sponges, hydrogels, fibers, etc. through processing technologies such as self-assembly, electrospinning, freeze drying and photocuring, showing a wide range of application potential in the field of biomedical engineering. Compared with other hemostatic materials, silk fibroin sponge can absorb water in the blood to form blood clots to block blood vessels. However, a single silk fibroin hemostatic sponge has problems such as insufficient hygroscopicity, hard texture, and local overfilling or insufficient filling. Hyaluronic acid is a natural polysaccharide polymer composed of D-glucuronic acid and N-acetylglucosamine. It is widely distributed in human connective tissue and has excellent biocompatibility and good biodegradability. Hyaluronic acid is often used in the study of hemostatic materials due to its high water retention, swelling and certain anti-adhesion effect. When the amount of bleeding from tissue wounds is large, the existing silk and / or hyaluronic acid hemostatic sponges are often unable to effectively achieve rapid hemostasis of the wound due to the single hemostatic method. At the same time, the lack of antibacterial and anti-inflammatory capabilities cannot meet actual clinical needs.

[0004] Therefore, developing a hemostatic material that can synergistically stop bleeding in multiple ways, has safe and long-lasting antibacterial ability, and has the potential to promote wound healing is of great significance for improving the effect of trauma treatment. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a multifunctional medical composite sponge and its preparation method and application. First, composite protein microspheres with long-term antibacterial, anti-inflammatory and synergistic hemostasis are prepared; then the composite protein microspheres are added to a mixed solution of silk fibroin, wool keratin and sodium hyaluronate, bubbles are generated by an oscillation cross-linking method, and the graded bubbles are stabilized by regulating the concentration and proportion of the mixed solution, and a multifunctional medical composite sponge (m-SF / WK / HA) with a graded pore structure is obtained after freeze drying. The m-SF / WK / HA prepared by the present invention has a fast and long-term liquid absorption ability, and the composite protein microspheres embedded in the m-SF / WK / HA release protamine and calcium ascorbate, enrich blood cells while having long-term antibacterial and anti-inflammatory effects, trigger a hemostatic cascade reaction, and achieve multiple hemostasis while having the potential to promote wound healing.

[0006] The first object of the present invention is to provide a method for preparing a multifunctional medical composite sponge, comprising the following steps:

[0007] S1, adding a first silk fibroin solution and ethanol to a first mixed solution, washing, and freeze-drying to obtain silk fibroin microspheres; the first mixed solution is obtained by dissolving calcium ascorbate and ascorbic acid in water;

[0008] S2, adding the silk fibroin microspheres described in S1 to the protamine solution, stirring, standing, centrifuging, and freeze-drying to obtain composite protein microspheres;

[0009] S3, after the second mixed solution is balanced at low temperature, a cross-linking agent and the composite protein microspheres described in S2 are added to carry out an oscillating cross-linking reaction, and then freeze-dried to obtain the multifunctional medical composite sponge; the second mixed solution is obtained by mixing a second silk fibroin solution, a wool keratin solution and a sodium hyaluronate solution, and the sodium hyaluronate is of medical grade.

[0010] In one embodiment of the present invention, in S1, the concentration of calcium ascorbate in the first mixed solution is 0.1 mg / L-10 mg / L, and the concentration of ascorbic acid is 0.1 g / L-3 g / L;

[0011] The concentration of silk fibroin in the first silk fibroin solution is 5 mg / mL-30 mg / mL;

[0012] The volume ratio of the first mixed solution to the first silk fibroin solution is 10:(1-5).

[0013] In one embodiment of the present invention, in S2, the concentration of protamine in the protamine solution is 5 mg / mL-20 mg / mL.

[0014] In one embodiment of the present invention, in S3, the concentration of silk fibroin in the second silk fibroin solution is 5 mg / mL-30 mg / mL, and the molecular weight of silk fibroin is 9 kDa-12 kDa; the concentration of wool keratin in the wool keratin solution is 2 mg / mL-8 mg / mL, and the molecular weight of wool keratin is 8 kDa-14 kDa; the concentration of sodium hyaluronate in the sodium hyaluronate solution is 5 mg / mL-10 mg / mL, and the molecular weight of sodium hyaluronate is 1000 kDa-2200 kDa;

[0015] The mass ratio of silk fibroin, wool keratin and sodium hyaluronate in the second mixed solution is (1-10):(0.1-4):(1-5);

[0016] The mass ratio of the second silk fibroin to the composite protein microspheres in the second mixed solution is (1-20):(1-20).

[0017] In one embodiment of the present invention, in S3, the cross-linking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0018] In one embodiment of the present invention, in S3, the mass ratio of silk fibroin, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the second mixed solution is (5-15):(2-6):(4-12).

[0019] In one embodiment of the present invention, in S3, the low temperature equilibrium is firstly standing at 0°C-4°C for 5min-10min, and then stirring at 20r / min-60r / min for 30s-600s.

[0020] In one embodiment of the present invention, in S3, the oscillating cross-linking reaction is first stirred at 50r / min-70r / min for 120s-240s, then oscillated at a speed of 60n / min-90n / min and an inclination angle of 45°-90° for 30s-60s, and allowed to stand at 0°C-4°C for 5s-10s; then oscillated at a speed of 30n / min-60n / min and an inclination angle of 45°-90° for 10s-30s, and allowed to stand at 0°C-4°C for 3s-5s;

[0021] The freeze drying is firstly freezing at -45°C to -35°C for 6h-8h, freezing at -85°C to -75°C for 10h-12h, and then drying for 48h-72h.

[0022] The second object of the present invention is to provide a multifunctional medical composite sponge prepared by the method described above.

[0023] The third object of the present invention is to provide an application of the multifunctional medical composite sponge in the preparation of hemostatic materials.

[0024] The technical solution of the present invention has the following advantages over the prior art:

[0025] (1) The multifunctional medical composite sponge (m-SF / WK / HA) prepared by the oscillation cross-linking method of the present invention has a simple and environmentally friendly preparation method. The prepared m-SF / WK / HA has a multi-graded pore structure and a high porosity, which promotes blood absorption and penetration while increasing the contact surface between the material and the blood, thereby increasing the blood absorption rate. HA and WK synergistically absorb moisture and swell to fix the liquid, showing a long-term liquid absorption capacity. After the liquid absorption and expansion, the internal capillary space is blocked, which is conducive to retaining blood and preventing blood leakage. The multi-graded and directional pore structure is conducive to the absorption of wound exudate and oxygen penetration while quickly absorbing liquid, avoiding wound surface fluid and cell hypoxia.

[0026] (2) The multifunctional medical composite sponge m-SF / WK / HA of the present invention is loaded with composite silk fibroin microspheres with long-term antibacterial, anti-inflammatory and synergistic hemostasis. The composite silk fibroin microspheres have uniform particle size, regular shape and porous surface, which is conducive to the slow and long-term release of drugs. The sustained release of protamine and calcium ascorbate not only plays a long-term antibacterial and anti-inflammatory role, but also helps to aggregate red blood cells, accelerate blood coagulation, and help enhance multiple hemostatic effects.

[0027] (3) The multifunctional medical composite sponge (m-SF / WK / HA) of the present invention has the dual hydrophilicity of HA and WK to enhance the liquid absorption capacity. The alkaline amino groups in the scaffold help neutralize the acidic substances on the wound surface and change the microenvironment of the wound surface. SF and WK promote cell growth, HA helps to reduce postoperative swelling and inflammation, and calcium ascorbate has antioxidant and anti-inflammatory effects to enhance the body's immunity, which multiple enhances the potential of m-SF / WK / HA to promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 The SEM images of the composite protein microspheres in Test Example 1 of the present invention; wherein (A) is Sample Example a, and (B) is Sample Example d;

[0030] Figure 2 This is the release curve of protamine in the composite protein microspheres in Test Example 2 of the present invention;

[0031] Figure 3 The SEM images of the composite sponge in Test Example 4 of the present invention; wherein (A), (B), (C), and (D) are sample 1 of Example 1, and (E) is comparative example 1;

[0032] Figure 4 The liquid absorption capacity of the multifunctional medical composite sponge in Test Example 5 of the present invention; wherein (A) is the liquid absorption rate of the sample after absorbing liquid for 1 minute, (B) is the liquid absorption rate of sample 1 at different times, and (C) is a naked eye observation photo of sample 1 after absorbing water and blood;

[0033] Figure 5 The diagram is a diagram of the in vitro procoagulant effect of the multifunctional medical composite sponge in Test Example 6 of the present invention; wherein (A) is a naked eye observation photograph of the in vitro procoagulant effect of Sample 1, (B) is a naked eye observation photograph of the in vitro procoagulant effect of Comparative Example 1, and (C) is a BCI bar graph of the sample at 1 min;

[0034] Figure 6 The multifunctional medical composite sponge in Test Example 7 of the present invention is attached to a tilted substrate after absorbing blood; wherein (A) is a photo just after tilting, and (B) is a photo of the sponge attached 30 seconds after tilting;

[0035] Figure 7 The antibacterial effect of the multifunctional medical composite sponge on Staphylococcus aureus in Test Example 8 of the present invention;

[0036] Figure 8 The results of the cell safety test of the multifunctional medical composite sponge on L929 cells in Test Example 9 of the present invention;

[0037] Fig. 9 This is the test result of the multifunctional medical composite sponge on the cell proliferation of L929 cells in Test Example 10 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] Example 1

[0040] The multifunctional medical composite sponge and the preparation method thereof of the present invention specifically comprise the following steps:

[0041] S1. Preparation of silk fibroin microspheres

[0042] S11. Preparation of silk fibroin solution: degumming silk with 0.5% papain solution at 100±1℃ for 95±1min (bath ratio 1:20), washing with deionized water for 4 times, and drying at 55±1℃ for 7h to obtain degummed silk; taking 15g of degummed silk, adding 100mL 9.3mol / L lithium bromide solution, dissolving at 60±1℃ for 5h, and after sufficient cooling, loading into a dialysis bag with a molecular weight cutoff of 9kDa-12kDa, dialyzing in deionized water at low temperature for 3d, changing the water every 5h; adjusting with deionized water after dialysis and ultrafiltration to obtain silk fibroin solutions with silk fibroin concentrations of 5mg / mL-20mg / mL.

[0043] S12, preparation of a mixed solution: dissolving calcium ascorbate in freshly boiled cold distilled water to obtain a calcium ascorbate solution, then dropping ascorbic acid into the calcium ascorbate solution, protecting from light, and stirring at 60 n / min for 60 s in an ice bath to obtain a mixed solution having a calcium ascorbate concentration of 1 mg / L-7 mg / L and an ascorbic acid concentration of 0.5 g / L-2 g / L;

[0044] S13, preparation of silk fibroin microspheres: drop 10 mL of 20 mg / mL silk fibroin solution into 100 mL of the mixed solution, vortex at a low speed of 60 n / min for 60 s, stand for 2 min in the dark, centrifuge at 1500 r / min for 3 min, discard the supernatant, add 1 mL-5 mL of ethanol, wash with ultrapure water for several times, and freeze-dry to obtain silk fibroin microspheres;

[0045] S2. Preparation of composite protein microspheres

[0046] S21, preparation of a protamine solution: dissolving protamine sulfate in ultrapure water to obtain a protamine solution having a protamine concentration of 5 mg / mL-20 mg / mL;

[0047] S22. Preparation of composite protein microspheres: adding silk fibroin microspheres to protamine solution, vortexing and stirring for 4 minutes, standing for 20 minutes, centrifuging at 1500 rpm for 3 minutes, discarding the supernatant, and freeze-drying to obtain composite protein microspheres assembled layer by layer;

[0048] Among them, the raw materials of the composite protein microspheres are shown in Table 1:

[0049] Table 1

[0050]

[0051] S3. Preparation of multifunctional medical composite sponge

[0052] S31. Preparation of wool keratin solution: take 5g of wool fiber, wash with ultrapure water and dry at 55±1℃; take the dried wool and add it into a mixed solution of ethanol / acetone with a volume ratio of 1:1 for 48h degreasing treatment, wash and dry to obtain degreased wool fiber; take 10g of degreased wool fiber and immerse it in 100mL reducing solution (sodium dodecyl sulfate: sodium sulfide: urea mass ratio = 1:3:42), stir and heat in a water bath at 50±1℃ for 9h, then put the mixed solution into a 8kDa-14kDa dialysis bag, dialyze with deionized water for 7d, change the deionized water every 6h, adjust with deionized water to obtain a wool keratin solution with a wool keratin concentration of 2mg / mL-8mg / mL;

[0053] S32, preparation of sodium hyaluronate solution: dissolving sodium hyaluronate powder with a molecular weight of 2100 kDa in deionized water to obtain a sodium hyaluronate solution with a sodium hyaluronate concentration of 5 mg / L-10 mg / mL;

[0054] S33, preparation of SF / WK / HA mixed solution: blend a silk fibroin solution with a concentration of 5 mg / mL-15 mg / mL and a wool keratin solution, stir at 30 r / min for 60 s in an ice bath, add the sodium hyaluronate solution, and continue stirring at 30 r / min for 120 s to obtain a SF / WK / HA mixed solution;

[0055] S34. Preparation of multifunctional medical composite sponge: The SF / WK / HA mixed solution was allowed to stand for 10 minutes under ice bath conditions, stirred at 30 r / min for 120 seconds, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added in sequence, stirred at 60 r / min for 30 minutes, the composite protein microspheres (sample example a) were added to the SF / WK / HA mixed solution, stirred at 60 r / min for 120 seconds, oscillated at a speed of 90 n / min and a tilt angle of 60° for 45 seconds, and allowed to stand for 5 seconds in an ice bath; then oscillated at a speed of 60 n / min and a tilt angle of 60° for 10 seconds, and allowed to stand for 5 seconds in an ice bath. The mixture was frozen at -40°C for 6 hours, frozen at -80°C for 12 hours, and dried for 48 hours to obtain a multifunctional medical composite sponge (m-SF / WK / HA).

[0056] Among them, the raw materials (mg / mL) of the multifunctional medical composite sponge are shown in Table 2, wherein Sample 4-Sample 5 are control examples:

[0057] Table 2

[0058]

[0059]

[0060] Comparative Example 1

[0061] The method is basically the same as Example 1, except that the preparation of the medical composite sponge specifically includes the following steps:

[0062] The SF solution was mixed with the HA solution, equilibrated in an ice bath for 10 minutes, and then stirred at 30 r / min for 120 seconds. N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added in sequence, and stirred at 60 r / min for 30 minutes. Then, the mixture was frozen at -40°C for 6 hours and dried for 48 hours to obtain a SF / HA medical composite sponge without a hierarchical pore structure.

[0063] Test Example 1

[0064] Based on Example 1, the composite protein microspheres (sample example a and sample example d) were characterized by SEM. The results are as follows Figure 1 As shown. Figure 1 It can be seen that the composite protein microspheres are quasi-circular, and the surface is rich in nanopore structures, which is conducive to the loading and release of drugs. The protamine molecules are assembled with the silk protein microspheres through electrostatic interaction, and calcium ascorbate is inside the microspheres. The dual system works together to exert long-term antibacterial, anti-inflammatory and hemostatic effects. As the protamine content increases, the particle size of the microspheres increases. Ethanol stabilizes the structure of silk microspheres and improves the antibacterial and anti-inflammatory effects. Calcium ascorbate has the effects of anti-oxidation and anti-inflammatory, enhancing the body's immunity, and can assist in activating platelets and coagulation cascade reactions, while anti-inflammatory and coordinating the coagulation mechanism, and has the potential to improve the hemostatic effect.

[0065] Test Example 2

[0066] Based on Example 1, a release experiment was conducted on the composite protein microspheres (sample example a and sample example d). 50 mg of composite protein microspheres were weighed and completely immersed in 10 mL of PBS solution, and slowly stirred at 25°C. 20 μL of solution was extracted from the tube after immersion for 10 min, 20 min, 30 min, 60 min, 120 min, 180 min and 240 min, and the supernatant was taken and filtered through a filter membrane with a pore size of 0.22 μm. The peak at 214 nm was measured by high performance liquid chromatography, and the protamine release curve of the composite protein microspheres after immersion for different time periods was plotted. The results are as follows: Figure 2 As shown. Figure 2It can be seen that in the first 30 minutes, there is a rapid release of protamine in the composite protein microspheres (sample example a), mainly because of the physical force between silk fibroin and the protamine on its surface, which causes the release of protamine on the surface of the composite protein microspheres. And the samples with a large amount of protamine added also have a large release amount (sample example d). With the increase of release time, the cumulative release of protamine in the composite protein microspheres gradually increases. After 180 minutes, the release curve of protamine in the composite protein microspheres tends to be stable, the release rate slows down, and a sustained release phenomenon is shown. It shows that the protamine in the composite protein microspheres can be slowly released, which helps to exert an antibacterial effect for a long time during the hemostasis and healing process of the wound. At the same time, protamine has coagulation activity, can activate the release of fibrinogen receptors and α particles, mediate platelet aggregation, and can assist in enhancing the coagulation effect.

[0067] Test Example 3

[0068] (1) Based on Example 1, the pore size distribution (%) of the multifunctional medical composite sponge (sample A-sample F) was analyzed, and the results are shown in Table 3:

[0069] Table 3

[0070] Sample Sample A Sample B Sample C Sample D Sample E Sample F <400μm 63.8±3.5 79.1±2.9 85.1±4.5 47.6±4.7 82.1±5.9 78.3±2.9 400μm-800μm 17.9±2.7 19.3±5.7 10.7±4.9 42.2±5.3 15.3±4.7 13.4±4.9 >800μm 18.3±3.1 1.6±3.2 4.2±0.9 10.2±2.8 2.6±1.1 8.3±1.2

[0071] It can be seen from Table 3 that under the same conditions, increasing the concentration of silk fibroin is beneficial to increasing the proportion of small pores (samples A-sample C), and increasing the concentration of sodium hyaluronate is beneficial to increasing the proportion of mesopores (samples A and D). By regulating the concentration and ratio of silk fibroin and sodium hyaluronate, the uniformity of the pore structure of m-SF / WK / HA is improved, and the bubbles in the m-SF / WK / HA mixed solution are stabilized, which is beneficial to increase the mutual penetration entanglement between the hierarchical pore layers.

[0072] (2) Based on Example 1, the pore size and porosity of the multifunctional medical composite sponge (Sample 1-Sample 5) were analyzed. The results are shown in Table 4:

[0073] Table 4

[0074]

[0075]

[0076] It can be seen from Table 4 that when wool keratin is not added, the pore size of m-SF / WK / HA is 271.6±62.4μm and the porosity is 84.7±4.6% (Sample 4). When the WK ratio is increased, the pore size and porosity of m-SF / WK / HA increase (Sample 1-Sample 2). However, when the addition amount of wool keratin is 10:7.5:10, the porosity of the m-SF / WK / HA antibacterial hemostatic sponge decreases slightly, which is caused by the partial collapse of the pore structure (Sample 3). Comparing the pore structures of Sample 1 and Sample 5, it can be seen that the addition of composite protein microspheres has no obvious effect on the pore size and porosity of m-SF / WK / HA.

[0077] Test Example 4

[0078] Based on Example 1 (Sample 1) and Comparative Example 1, the composite sponge was characterized by SEM. The results are as follows Figure 3 As shown. Figure 3 The top view of (A) and Figure 3 As can be seen from the side-section view in (B), the embodiment increases the bubbles in the SF / WK / HA mixed solution by the oscillation cross-linking method to form a multifunctional medical composite sponge (m-SF / WK / HA) with a loose upper part and a dense lower part with a multi-graded pore structure. Figure 3 From the cross-section of (C), we can see that the internal structure of m-SF / WK / HA is a connected multi-level pore structure with certain orientation, which helps the composite sponge to conduct liquid in a single direction and achieve rapid blood absorption. The internal structure of m-SF / WK / HA is loaded with long-acting antibacterial composite protein microspheres ( Figure 3 (D)) releases protamine, which facilitates cell migration and the exchange of nutrients and metabolites during tissue repair. Figure 3 As can be seen in (E), the medical composite sponge (SF / HA) prepared in the comparative example without the oscillation cross-linking method does not have a hierarchical pore structure and is not directional.

[0079] Test Example 5

[0080] Based on Example 1, the liquid absorption capacity of the multifunctional medical composite sponge (samples 1-3, 5 and comparative example 1) was tested. Accurately weighed and recorded as W dry , soaked in physiological saline and anticoagulated mouse whole blood for 1 min, 3 min, 5 min and 60 min at room temperature, taken out, and placed on a 60° inclined glass for 1 min, wiped off the excess liquid around the material, and weighed again, recorded as W wet , calculate the liquid absorption rate of m-SF / WK / HA to determine its liquid absorption capacity. Liquid absorption rate = (W wet -W dry ) / W dry × 100%, the result is as follows Figure 4 As shown. Figure 4It can be seen from (A) that after absorbing whole blood for 1 minute, the liquid absorption rate of sample 1 is 1772.06±28.99%, which is higher than the liquid absorption rate of comparative example 1 of 1307.01±31.29%, indicating that m-SF / WK / HA has a higher liquid absorption capacity, which is related to the multi-layered porous structure of m-SF / WK / HA and the increase of WK. The liquid absorption rate of sample 3 is 2394.37±52.78%, indicating that increasing the wool keratin content can increase the liquid absorption rate of m-SF / WK / HA. The liquid absorption rate of sample 1 is slightly higher than that of sample 5 (1668.43±29.31%), indicating that the addition of composite protein microspheres helps to improve the liquid absorption capacity of m-SF / WK / HA. Under the same conditions, the absorption rate of sample 1 for saline was 1827.78±30.52%, slightly higher than its absorption rate for whole blood (1772.06±28.99%). This was mainly because the protamine on the composite protein microspheres loaded in m-SF / WK / HA promoted the attraction of m-SF / WK / HA to anionic coagulation substances such as red blood cells and platelets, accelerated the aggregation of red blood cells at the junction of m-SF / WK / HA and the wound surface, and formed blood clots inside the material, resulting in a slight decrease in the absorption volume. Figure 4 As can be seen in (B), the absorption rate of blood by sample 1 after immersion for 5 minutes was 2003.31±86.34%, and the absorption rate of saline was 2207.21±77.01%, and the absorption amount reached the maximum, indicating that m-SF / WK / HA has the ability to absorb liquid quickly, which is conducive to achieving rapid hemostasis. After 60 minutes of absorption, m-SF / WK / HA gradually reached equilibrium, showing long-term hemostatic potential.

[0081] Test Example 6

[0082] Based on Example 1, the in vitro coagulation performance of the multifunctional medical composite sponge (samples 1-3, 5 and comparative example 1) was tested. 0.2 mL of fresh anticoagulated mouse whole blood was added to sample 1, and 0.02 mL of CaCl 2 Solution (0.1 mol / L), incubate for 5 min, add 25 mL of deionized water, shake for 5 min, and measure the absorbance at 545 nm using a spectrophotometer, recorded as A 样品 ; The absorbance measured by adding 0.2 mL of fresh anticoagulated mouse whole blood to 25 mL of deionized water is recorded as A blank; calculate the BCI of m-SF / WK / HA, BCI = A 样品 / A 空白 × 100%, the result is as follows Figure 5 As shown. Figure 5 Middle (A) and Figure 5In (B), it can be seen that sample 1 turns red after adding whole blood, with blood clots on the surface. After soaking in deionized water, no obvious red blood cells are dissolved around it, while comparative example 1 has red blood cells dissolved around it after soaking in deionized water. Figure 5 It can be seen from (C) that at 1 min, the BCI of sample 1 is 4.79±0.59%, which is significantly lower than that of comparative example 1 (20.34±2.46%), indicating that m-SF / WK / HA has better hemostatic and coagulation ability. On the one hand, due to the dual hydrophilicity of HA and WK and the stable hierarchical pore structure of m-SF / WK / HA, the liquid absorption speed is increased, blood viscosity is promoted, and exogenous coagulation ability is improved. On the other hand, the composite protein microspheres loaded in m-SF / WK / HA promote the adhesion and aggregation of platelets and accelerate endogenous blood coagulation to achieve multiple hemostatic purposes. When the WK content in m-SF / WK / HA increases, the BCI value decreases slightly, indicating that the hemostatic performance of m-SF / WK / HA is slightly improved, which is related to the improvement of the pore structure and liquid absorption capacity. Comparing the BCI of sample 1 and sample 5, it can be seen that the addition of composite protein microspheres is beneficial to reducing the BCI of m-SF / WK / HA, which is related to the release of calcium ascorbate and protamine in the composite protein microspheres. 2+ It can bind to platelet receptors, promote platelet adhesion and aggregation, and help start the endogenous hemostasis mechanism. In addition, calcium ascorbate has antioxidant and anti-inflammatory effects, helps the synthesis of collagen, and promotes wound healing. The surface of protamine in the composite protein microspheres is positively charged, which is beneficial to promote the aggregation of red blood cells and accelerate blood coagulation. Compared with the previous silk protein / hyaluronic acid composite scaffolds, the m-SF / WK / HA multifunctional medical composite sponge, based on the dual hydrophilicity of HA and WK, the alkaline amino groups in the scaffold help neutralize the acidic substances on the wound surface and change the microenvironment of the wound surface. In summary, m-SF / WK / HA has multiple hemostatic and wound protection capabilities and promotes wound healing.

[0083] Test Example 7

[0084] The multifunctional medical composite sponge (sample 1 and comparative example 1) after absorbing blood was placed on cellulose weighing paper, and the adhesion ability of the material was observed with the naked eye after tilting at 60° for 30 seconds. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that both sample 1 and comparative example 1 can adhere to the substrate at first (A). After 30 seconds, compared with comparative example 1 sliding off the paper surface after absorbing blood, sample 1 can stay on the paper surface (B), indicating that m-SF / WK / HA has a certain adhesion ability, which is conducive to staying on the wound surface and better playing the role of absorbing blood and stopping bleeding.

[0085] Test Case 8

[0086] Based on Example 1, the antibacterial ability of the multifunctional medical composite sponge (Sample 1-Sample 3 and Comparative Example 1) was tested by oscillation method. 0.5 g of the multifunctional medical composite sponge was weighed, cut into pieces, and sterilized with ultraviolet light for later use. The Staphylococcus aureus liquid was diluted to 10% with sterile PBS solution. 5 CFU / mL, take 50mL of diluted bacterial solution and add it to the sample, shake and culture it in a constant temperature oscillator at 37℃ for 1 day, 3 days and 5 days, take 100μL of bacterial solution and evenly spread it on LB solid culture medium, culture it in a constant temperature incubator at 37℃ for 24h, count the number of colonies and calculate the antibacterial rate. Antibacterial rate = (1-experimental bacterial solution concentration / blank bacterial solution concentration) × 100%, the results are as follows Figure 7 As shown. Figure 7 It can be seen that after 1 day, the antibacterial rate of sample 1 against Staphylococcus aureus was 77.34±4.31%, indicating that the loaded silk fibroin microspheres release antibacterial agents outward. With the increase of the loading amount of the composite protein microspheres, the antibacterial ability of m-SF / WK / HA increased (sample 3 reached 96.42±5.21%), while comparative example 1 had no obvious antibacterial ability. From the statistical results of the antibacterial rate on different days, the composite protein microspheres loaded by m-SF / WK / HA can release antibacterial agents for a long time, and the released protamine can inhibit the adhesion bacteria of Staphylococcus aureus biofilm to produce extracellular polysaccharides, showing a long-term antibacterial effect, which helps to prevent bleeding wound infection and promote wound repair.

[0087] Test Example 9

[0088] Based on Example 1, the toxicity of the multifunctional medical composite sponge to L929 mouse fibroblasts was detected with reference to the national standard GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test". After extracting m-SF / WK / HA (sample 1-sample 3 and comparative example 1) with DMEM containing 10% fetal bovine serum for 72 hours, the extract was filtered through a 0.22μm microporous filter membrane and the cells were cultured. After culturing for 24 hours, the original culture medium was discarded, and DMEM containing 10% CCK-8 was added to the cells and incubated for 2 hours. After shaking, the absorbance value was measured at 450nm on the microplate reader. The formula was used to calculate the cell viability to determine the effect of m-SF / WK / HA on the proliferation of L929 cells. Cell viability (%) = (OD tes -OD pos ) / (OD neg -OD pos )×100. Among them, OD tes Indicates the absorbance of the test sample, OD pos Represents the absorbance of the positive control group (cytotoxicity reached 100%), OD neg represents the absorbance of the negative control group (the control group with only cell culture medium). Figure 8As shown. Figure 8 It can be seen that after 24 h, the cell survival rate of each group of samples was above 80%, indicating that m-SF / WK / HA has good cell safety.

[0089] Test Example 10

[0090] Based on Example 1, the proliferation activity of L929 cells on the m-SF / WK / HA composite medical sponge was detected by CCK-8 method. L929 cells were planted on the m-SF / WK / HA composite medical sponge (sample 1-sample 3 and comparative example 1), and the culture medium was discarded after culturing for 1d, 3d and 5d, respectively. After rinsing with PBS solution, DMEM containing 10% CCK-8 was added and incubated for 2h. After shaking, the absorbance value was measured at 450nm on the microplate reader. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that with the increase of the number of culture days, the OD value of each group of materials increased, indicating that each group of m-SF / WK / HA composite medical sponges can support the growth and proliferation of cells. The OD values ​​of samples 1 to 3 are not significantly different from the OD values ​​of comparative example 1, indicating that the composite protein microspheres loaded by m-SF / WK / HA have no significant effect on cell growth, indicating that the m-SF / WK / HA composite medical sponge can support cell proliferation while having antibacterial ability and has the potential to promote wound healing.

[0091] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a multifunctional medical composite sponge, characterized in that: The following steps are involved: S1, adding a first silk fibroin solution and ethanol to a first mixed solution, washing, and freeze-drying to obtain silk fibroin microspheres; the first mixed solution is obtained by dissolving calcium ascorbate and ascorbic acid in water; S2, adding the silk fibroin microspheres described in S1 to the protamine solution, stirring, standing, centrifuging, and freeze-drying to obtain composite protein microspheres; S3, after the second mixed solution is low temperature balanced, add the cross-linking agent and the composite protein microspheres described in S2, perform an oscillation cross-linking reaction, and then freeze-dry to obtain the multifunctional medical composite sponge; The second mixed solution is obtained by mixing a second silk fibroin solution, a wool keratin solution and a sodium hyaluronate solution; the low temperature equilibrium is firstly standing at 0°C-4°C for 5min-10min, and then stirring at 20r / min-60r / min for 30s-600s.

2. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S1, the concentration of calcium ascorbate in the first mixed solution is 0.1 mg / L-10 mg / L, and the concentration of ascorbic acid is 0.1 g / L-3 g / L; The concentration of silk fibroin in the first silk fibroin solution is 5 mg / mL-30 mg / mL; The volume ratio of the first mixed solution to the first silk fibroin solution is 10:(1-5).

3. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S2, the concentration of protamine in the protamine solution is 5 mg / mL-20 mg / mL.

4. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S3, the concentration of silk fibroin in the second silk fibroin solution is 5 mg / mL-30 mg / mL, and the molecular weight of silk fibroin is 9 kDa-12 kDa; the concentration of wool keratin in the wool keratin solution is 2 mg / mL-8 mg / mL, and the molecular weight of wool keratin is 8 kDa-14 kDa; the concentration of sodium hyaluronate in the sodium hyaluronate solution is 5 mg / mL-10 mg / mL, and the molecular weight of sodium hyaluronate is 1000 kDa-2200 kDa; The mass ratio of silk fibroin, wool keratin and sodium hyaluronate in the second mixed solution is (1-10): (0.1-4): (1-5); The mass ratio of the second silk fibroin to the composite protein microspheres in the second mixed solution is (1-20): (1-20).

5. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S3, the cross-linking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

6. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S3, the mass ratio of silk fibroin, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the second mixed solution is (5-15):(2-6):(4-12).

7. The method for preparing the multifunctional medical composite sponge according to claim 1, characterized in that: In S3, the oscillation cross-linking reaction is first stirred at 50r / min-70r / min for 120s-240s, then oscillated at a speed of 60n / min-90n / min and an inclination angle of 45°-90° for 30s-60s, and allowed to stand at 0°C-4°C for 5s-10s; then oscillated at a speed of 30n / min-60n / min and an inclination angle of 45°-90° for 10s-30s, and allowed to stand at 0°C-4°C for 3s-5s; The freeze drying is firstly freezing at -45°C to -35°C for 6h-8h, freezing at -85°C to -75°C for 10h-12h, and then drying for 48h-72h.

8. A multifunctional medical composite sponge prepared by the method according to any one of claims 1 to 7.

9. Use of the multifunctional medical composite sponge as claimed in claim 8 in preparing hemostatic materials.

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