CN119258260B - Multifunctional medical composite sponge and preparation method and application thereof - Google Patents

Abstract

The invention relates to a multifunctional medical composite sponge and a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the steps of adding a first silk fibroin solution and ethanol into a first mixed solution, washing and freeze-drying to obtain silk fibroin microspheres, wherein the first mixed solution is prepared by dissolving calcium ascorbate and ascorbic acid into water, adding silk fibroin microspheres into a protamine solution, stirring, standing, centrifuging and freeze-drying to obtain composite protein microspheres, adding a cross-linking agent and the composite protein microspheres for oscillation cross-linking reaction after the second mixed solution is balanced at low temperature, and freeze-drying to obtain the multifunctional medical composite sponge, wherein the second mixed solution is prepared by mixing a second silk fibroin solution, a wool keratin solution and a sodium hyaluronate solution. The sponge can induce hemostatic cascade reaction while absorbing liquid for a long time, resisting bacteria and diminishing inflammation, realizes multiple hemostasis and has the potential of promoting wound healing.

Description

Multifunctional medical composite sponge and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a multifunctional medical composite sponge and a preparation method and application thereof.

Background

In a daily life scenario, it is a common and difficult to completely circumvent phenomenon that skin or tissue is subjected to trauma and associated bleeding. When the wound reaches a large range or depth, the human body may be rapidly caused to lose blood too much, and thus the life is seriously threatened. If an uncontrolled bleeding situation is not effectively controlled within the critical thirty minutes of emergency treatment, the risk of injury and death will rise dramatically. Therefore, the development of a novel hemostatic material capable of rapidly responding and realizing hemostasis in multiple ways has become a core trend of the development of the future hemostatic technology. The ideal hemostatic material has the potential of rapidly and efficiently controlling bleeding, long-acting antibacterial capacity, biodegradability, good biocompatibility and promoting wound healing.

The porous sponge is used as a repair material with wide application, and the porous sponge has a large number of pore structures, so that the porous sponge is favorable for promoting absorption of body fluid and exchange of tissue fluid, and plays an important role in the field of hemostasis treatment. The silk fibroin is natural high molecular protein extracted from silk, and has good biocompatibility and controllable biodegradability. Silk fibroin can be converted into materials with various forms such as nano particles, films, sponges, hydrogels, fibers and the like through processing technologies such as self-assembly, electrostatic spinning, freeze drying, photo-curing and the like, and has wide application potential in the field of biomedical engineering. Compared with other hemostatic materials, the silk fibroin sponge can absorb moisture in blood, and form scabs to block blood vessels. However, single silk fibroin hemostatic sponge has the problems of insufficient hygroscopicity, harder texture, partial overfilling or insufficient filling and the like. Hyaluronic acid is a natural polysaccharide polymer composed of D-glucuronic acid and N-acetylglucosamine, is widely distributed in connective tissue of human body, and has excellent biocompatibility and good biodegradability. Hyaluronic acid is often used for research on hemostatic materials because of its high water-retaining property, swelling property and certain anti-blocking effect. When the bleeding amount of the tissue wound is large, the existing silk fibroin or/and hyaluronic acid hemostatic sponge cannot effectively realize rapid hemostasis of the wound due to a single hemostatic mode, and meanwhile, the lack of antibacterial and anti-inflammatory capabilities cannot meet the actual clinical requirements.

Therefore, a hemostatic material which can be used for rapidly stopping bleeding in a synergistic way and has the functions of improving the antibacterial capacity of the whole body and promoting the healing potential of wounds is developed, and has important significance for improving the wound treatment effect.

Disclosure of Invention

In order to solve the technical problems, the invention provides a multifunctional medical composite sponge and a preparation method and application thereof. Firstly preparing long-acting antibacterial anti-inflammatory synergistic hemostatic composite protein microspheres, then adding the composite protein microspheres into a mixed solution of silk fibroin, wool keratin and sodium hyaluronate, generating bubbles by an oscillation crosslinking method, stabilizing and grading the bubbles by regulating and controlling the concentration and the proportion of the mixed solution, and freeze-drying to obtain the multifunctional medical composite sponge (m-SF/WK/HA) with a graded pore structure. The m-SF/WK/HA prepared by the invention HAs the capacity of rapid long-acting liquid absorption, and simultaneously, the composite protein microspheres embedded in the m-SF/WK/HA release protamine and calcium ascorbate, enrich blood cells while resisting bacteria and diminishing inflammation for a long time, initiate a hemostatic cascade reaction, realize multiple hemostasis and simultaneously have the potential of promoting wound healing.

The first object of the invention is to provide a preparation method of a multifunctional medical composite sponge, which comprises the following steps:

s1, adding a first silk fibroin solution and ethanol into a first mixed solution, and washing and freeze-drying to obtain silk fibroin microspheres, wherein the first mixed solution is prepared by dissolving calcium ascorbate and ascorbic acid in water;

s2, adding the silk fibroin microsphere described in the S1 into a protamine solution, stirring, standing, centrifuging and freeze-drying to obtain a composite protein microsphere;

S3, adding a cross-linking agent and the composite protein microsphere in the S2 after the second mixed solution is balanced at a low temperature, performing oscillation cross-linking reaction, and then performing freeze drying to obtain the multifunctional medical composite sponge, wherein 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 medical grade.

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

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

the volume ratio of the first mixed solution to the first silk fibroin solution is 10 (1-5).

In one embodiment of the invention, in S2, the concentration of protamine in the protamine solution is 5mg/mL to 20mg/mL.

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

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 microsphere in the second mixed solution is (1-20): 1-20.

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

In one embodiment of the 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.

In one embodiment of the present invention, in S3, the low temperature equilibration is first allowed to stand at 0-4℃for 5-10 min, then stirred at 20-60 r/min for 30S-600S.

In one embodiment of the invention, in S3, the oscillation crosslinking reaction is that firstly stirring for 120S-240S at 50r/min-70r/min, then oscillating for 30S-60S at a speed of 60n/min-90n/min and an inclination angle of 45-90 ℃, standing for 5S-10S at 0-4 ℃, and then oscillating for 10S-30S at a speed of 30n/min-60n/min and an inclination angle of 45-90 ℃ and standing for 3S-5S at 0-4 ℃;

The freeze drying is to freeze at-45 ℃ to-35 ℃ for 6h-8h, at-85 ℃ to-75 ℃ for 10h-12h, and then dry for 48h-72h.

The second object of the invention is to provide a multifunctional medical composite sponge prepared by the method.

The third object of the invention is to provide an application of the multifunctional medical composite sponge in preparing hemostatic materials.

Compared with the prior art, the technical scheme of the invention has the following advantages:

(1) The preparation method is simple and environment-friendly, and the prepared m-SF/WK/HA HAs a multi-hierarchical pore structure and higher porosity, so that the absorption and permeation of blood are promoted, the contact surface between the material and the blood is improved, and the absorption speed of the blood is improved. HA and WK cooperate with the hygroscopic expansion to fix liquid, and the liquid absorbing capacity is shown for a long time, and after the liquid absorbing expansion, the internal capillary space is blocked, so that the blood can be kept, and the blood leakage can be prevented. The multi-stage and directional pore structure is beneficial to absorption of wound exudates and oxygen permeation while absorbing liquid rapidly, and avoids wound area liquid and cell hypoxia.

(2) The multifunctional medical composite sponge m-SF/WK/HA is internally loaded with the long-acting antibacterial anti-inflammatory synergistic hemostatic composite silk fibroin microsphere. The composite silk fibroin microsphere has uniform particle size, regular shape and porous surface, and is beneficial to slow and long-acting release of the medicine. The slow release of protamine and calcium ascorbate plays a long-acting antibacterial and anti-inflammatory role, and simultaneously is beneficial to aggregating red blood cells, accelerating blood coagulation and assisting in improving multiple hemostatic effects.

(3) The multifunctional medical composite sponge (m-SF/WK/HA) disclosed by the invention is beneficial to neutralizing acidic substances of a wound surface on the basis of the double hydrophilia reinforced liquid absorption capacity of HA and WK, and the basic amino groups in the bracket are beneficial to changing the microenvironment of the wound surface. SF and WK promote cell growth, HA helps to reduce postoperative swelling and inflammation, calcium ascorbate resists oxidation and inflammation, enhances organism immunity, and multiple increases m-SF/WK/HA potential for promoting wound healing.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which:

FIG. 1 is an SEM image of composite protein microspheres of test example 1 according to the present invention, wherein (A) is sample example a and (B) is sample example d;

FIG. 2 is a graph showing the release profile of protamine from the composite protein microsphere of test example 2 according to the present invention;

FIG. 3 is an SEM image of a composite sponge of 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;

FIG. 4 is a graph showing the liquid absorption capacity of the multifunctional medical composite sponge according to test example 5 of the present invention, wherein (A) is the liquid absorption rate of the sample at 1min of liquid absorption, (B) is the liquid absorption rate of the sample 1 at different times, and (C) is a photograph of the sample 1 after water absorption and blood absorption by naked eyes;

FIG. 5 is a graph showing the in vitro procoagulant effect of the multifunctional medical composite sponge according to test example 6 of the present invention, wherein (A) is a visual photograph of the in vitro procoagulant effect of sample 1, (B) is a visual photograph of the in vitro procoagulant effect of comparative example 1, and (C) is a BCI histogram of the sample at 1 min;

FIG. 6 is a photograph showing adhesion of the multifunctional medical composite sponge of test example 7 of the present invention to an inclined substrate after blood suction, wherein (A) is a photograph immediately after inclination, and (B) is a photograph showing adhesion after inclination for 30 seconds;

FIG. 7 shows the bacteriostatic effect of the multifunctional medical composite sponge of test example 8 according to the invention on Staphylococcus aureus;

FIG. 8 is a graph showing the results of the cell safety test of the multifunctional medical composite sponge of test example 9 on L929 cells according to the present invention;

FIG. 9 shows the results of the cell proliferation test of L929 cells by the multifunctional medical composite sponge according to test example 10 of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1

The invention relates to a multifunctional medical composite sponge and a preparation method thereof, which specifically comprise the following steps:

S1, preparation of silk fibroin microspheres

S11, preparing a silk fibroin solution, namely degumming silk by using a papain solution with the mass fraction of 0.5% at the temperature of 100+/-1 ℃ for 95+/-1 min (bath ratio of 1:20), washing for 4 times by using deionized water, baking for 7 hours at the temperature of 55+/-1 ℃ to obtain degummed silk, adding 100mL of lithium bromide solution with the concentration of 9.3mol/L into 15g of degummed silk, dissolving for 5 hours at the temperature of 60+/-1 ℃, fully cooling, filling into a dialysis bag with the molecular weight cutoff of 9kDa-12kDa, dialyzing for 3 days in deionized water at low temperature, changing water every 5 hours, and regulating by using deionized water after dialysis and ultrafiltration to obtain the silk fibroin solution with the concentration of 5mg/mL-20mg/mL respectively.

S12, preparing a mixed solution, namely dissolving calcium ascorbate into cold distilled water which is boiled newly to obtain a calcium ascorbate solution, then dropwise adding ascorbic acid into the calcium ascorbate solution, and stirring for 60S under the condition of avoiding light and ice bath for 60n/min to obtain a mixed solution with the concentration of 1mg/L-7mg/L of calcium ascorbate and the concentration of 0.5g/L-2g/L of ascorbic acid;

s13, preparing silk fibroin microspheres, namely dripping 10mL of silk fibroin solution with the concentration of 20mg/mL into 100mL of mixed solution, carrying out low-speed vortex for 60S at 60n/min, standing for 2min in a dark place, centrifuging for 3min at 1500r/min, discarding the supernatant, adding 1mL-5mL of ethanol, washing with ultrapure water for multiple times, and freeze-drying to obtain the silk fibroin microspheres;

S2, preparation of composite protein microsphere

S21, preparing a protamine solution, namely dissolving protamine sulfate in ultrapure water to obtain a protamine solution with the concentration of 5-20 mg/mL;

S22, preparing composite protein microspheres, namely adding the silk fibroin microspheres into a protamine solution, performing vortex stirring for 4min, standing for 20min, centrifuging for 3min at 1500r/min, discarding the supernatant, and performing freeze drying to obtain composite protein microspheres assembled layer by layer;

Wherein, the raw materials of the composite protein microsphere are shown in table 1:

TABLE 1

S3, preparation of multifunctional medical composite sponge

S31, preparing a wool keratin solution, namely taking 5g of wool fibers, washing the wool fibers with ultrapure water, drying the wool fibers at 55+/-1 ℃, adding the dried wool fibers into a mixed solution of ethanol/acetone with the volume ratio of 1:1, degreasing the wool fibers for 48 hours, washing and drying the wool fibers to obtain degreased wool fibers, immersing 10g of degreased wool fibers into 100mL of reducing solution (sodium dodecyl sulfate: sodium sulfide: urea mass ratio=1:3:42), stirring and heating the degreased wool fibers in a water bath with the temperature of 50+/-1 ℃ for 9 hours, filling the mixed solution into a dialysis bag with 8kDa-14kDa, dialyzing the mixture for 7d with deionized water, changing the deionized water every 6 hours, and regulating the mixture with the deionized water to obtain the wool keratin solution with the concentration of 2mg/mL-8 mg/mL;

s32, preparing a sodium hyaluronate solution, namely dissolving sodium hyaluronate powder with the molecular weight of 2100kDa into deionized water to obtain sodium hyaluronate solutions with the sodium hyaluronate concentration of 5mg/L-10mg/mL respectively;

S33, preparing SF/WK/HA mixed solution, namely blending 5mg/mL-15mg/mL silk fibroin solution and wool keratin solution, stirring for 60S at 30r/min under ice bath condition, adding sodium hyaluronate solution, and continuously stirring for 120S at 30r/min to obtain SF/WK/HA mixed solution;

S34, preparing a multifunctional medical composite sponge, namely standing and balancing SF/WK/HA mixed solution for 10min under ice bath conditions, stirring for 120S at 30r/min, sequentially adding N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), stirring for 30min at 60r/min, adding composite protein microspheres (sample a) into the SF/WK/HA mixed solution, stirring for 120S at 60r/min, oscillating for 45S at a speed of 90N/min and an inclination angle of 60 degrees, standing for 5S in an ice bath, oscillating for 10S at a speed of 60N/min and an inclination angle of 60 degrees, and standing for 5S in an ice bath. Freezing at-40 ℃ for 6h and-80 ℃ for 12h, and drying for 48h to obtain the multifunctional medical composite sponge (m-SF/WK/HA).

Wherein, the raw materials (mg/mL) of the multifunctional medical composite sponge are shown in Table 2, and the samples 4-5 are comparative examples:

TABLE 2

Comparative example 1

Basically the same as example 1, the preparation of the medical composite sponge specifically comprises the following steps:

mixing SF solution and HA solution, balancing for 10min under ice bath condition, stirring for 120s at 30r/min, sequentially adding N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), stirring for reacting for 30min at 60r/min, freezing at-40 ℃ for 6h, and drying for 48h to obtain SF/HA medical composite sponge without hierarchical pore structure.

Test example 1

Based on example 1, SEM characterization was performed on composite protein microspheres (sample a and sample d), and the results are shown in fig. 1. From fig. 1, it can be seen that the composite protein microsphere is round-like, and the surface is rich in a nano-pore structure, which is helpful for loading and releasing drugs. The protamine molecules are assembled with the silk fibroin microspheres through electrostatic action, and calcium ascorbate is positioned in the microspheres, and the dual systems jointly act to play a role in long-acting antibiosis, antiphlogosis and hemostasis. The protamine content is increased, and the microsphere particle size is increased. Ethanol stabilizes the silk fibroin microsphere structure and improves the antibacterial and anti-inflammatory effects. The calcium ascorbate has the functions of resisting oxidation and inflammation, enhancing organism immunity, assisting in activating platelet and coagulation cascade reaction, and improving the potential of hemostatic effect by synergizing a coagulation mechanism while resisting inflammation.

Test example 2

Based on example 1, release experiments were performed on composite protein microspheres (sample a and sample d), 50mg of the composite protein microspheres were weighed and completely soaked in 10mLPBS solutions, respectively, and 20 μl of the solution was extracted from the tube at 25 ℃, at the time of soaking for 10min, 20min, 30min, 60min, 120min, 180min and 240min with slow stirring, centrifuged at 5000r/min for 3min, and the supernatant was filtered through a filter membrane with a pore size of 0.22 μm. The peak value at 214nm is measured by high performance liquid chromatography, and the protamine release curve of the composite protein microsphere after being soaked for different time is drawn, and the result is shown in figure 2. As can be seen from fig. 2, there is a rapid release of protamine within the composite protein microsphere (sample a) for the first 30min, mainly due to the physical forces existing between the silk fibroin and the protamine on its surface, which causes the release of protamine on the surface of the composite protein microsphere. In addition, the amount of protamine released was large in the sample with large amount of protamine added (sample d). The cumulative release of protamine in the composite protein microspheres gradually increases with increasing release time. After 180min, the release curve of protamine in the composite protein microsphere tends to be stable, the release speed is slow, and the slow release phenomenon is presented. The composite protein microsphere has the advantages that protamine in the composite protein microsphere can be slowly released, and the antibacterial effect can be exerted for a long time in the wound hemostasis healing process. Meanwhile, protamine has coagulation activity, can activate the release of fibrinogen receptor and alpha particles, mediate the aggregation of platelets, and can assist in enhancing the coagulation effect.

Test example 3

(1) Based on example 1, the pore size distribution (%) of the multifunctional medical composite sponge (sample a to sample F) was analyzed, and the results are shown in table 3:

TABLE 3 Table 3

Sample preparation

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

As can be seen from table 3, under the same conditions, increasing the concentration of silk fibroin favors the increase of the ratio of the pores (samples a-C), and increasing the concentration of sodium hyaluronate favors the increase of the ratio of the mesopores (samples a and D). The concentration and the proportion of the silk fibroin and the sodium hyaluronate are regulated to improve the uniformity of the m-SF/WK/HA pore structure, so that bubbles in the m-SF/WK/HA mixed solution are stabilized, and the mutual osmosis entanglement between the grading pore layers is facilitated to be increased.

(2) Based on example 1, pore size and porosity of the multifunctional medical composite sponge (sample 1-sample 5) were analyzed, and the results are shown in table 4:

TABLE 4 Table 4

As can be seen from Table 4, when wool keratin is not added, the pore size of m-SF/WK/HA is 271.6.+ -. 62.4. Mu.m, and the porosity is 84.7.+ -. 4.6% (sample 4). As the WK ratio was increased, the m-SF/WK/HA pore size and porosity increased (samples 1-2). However, when the wool keratin addition amount was 10:7.5:10, the porosity of the m-SF/WK/HA antibacterial hemostatic sponge was slightly decreased due to the partial collapse of the pore structure (sample 3). The pore structures of sample 1 and sample 5 can be compared, and it can be seen that the addition of the composite protein microsphere HAs no obvious effect on the pore diameter and porosity of m-SF/WK/HA.

Test example 4

Based on example 1 (sample 1) and comparative example 1, SEM characterization was performed on the composite sponge, and the results are shown in fig. 3. As can be seen from the top view of (A) in FIG. 3 and the side cut view of (B) in FIG. 3, the embodiment increases bubbles in SF/WK/HA mixed solution by an oscillation crosslinking method to form the multifunctional medical composite sponge (m-SF/WK/HA) with an upper loose lower compact multi-stage pore structure. As can be seen from the transverse drawing of the figure 3 (C), the m-SF/WK/HA HAs a multi-stage pore structure with communicated inside, HAs certain directionality, and is beneficial to the compound sponge to carry out one-way liquid guiding and realize rapid blood suction. The m-SF/WK/HA is internally loaded with the long-acting antibacterial composite protein microsphere (shown in (D) in fig. 3), so that the protamine is released, and meanwhile, the migration of cells in the tissue repair process and the exchange of nutrients and metabolites are facilitated. As can be seen from fig. 3 (E), the medical composite sponge (SF/HA) prepared in the comparative example, which was not prepared by the oscillation crosslinking method, HAs no hierarchical pore structure and HAs no directionality.

Test example 5

Based on example 1, the liquid absorbing ability of the multifunctional medical composite sponges (samples 1 to 3, 5 and comparative example 1) was tested. Accurately weighing, recording as W _dry, soaking in normal saline and anticoagulated mouse whole blood at room temperature for 1min, 3min, 5min and 60min, taking out, standing on 60-degree inclined glass for 1min, wiping off redundant liquid around the material, weighing again, recording as W _wet, calculating the liquid absorption rate of m-SF/WK/HA, and judging the liquid absorption capacity. As can be seen from FIG. 4 (A), the liquid absorption= (W _wet-W_dry)/W_dry X100%, the result is shown in FIG. 4. The liquid absorption rate of sample 1 is 1772.06 + -28.99% higher than that of comparative example 1, indicating that the liquid absorption capacity of sample 1 to physiological saline is higher than that of 1307.01 + -31.29%, which is related to the multi-layered pore structure of m-SF/WK/HA and the increase of WK. The liquid absorption rate of sample 3 is 2394.37 + -52.78%, which indicates that the liquid absorption rate of sample 1 can be increased by increasing the keratin content of m-SF/WK/HA, which is slightly higher than that of sample 5 (1668.43 + -29.31%), indicating that the liquid absorption rate of sample 1 to physiological saline is 1827.78 + -30.52% higher than that of sample 1 to physiological saline, which is mainly because of the liquid absorption rate of m-SF/SF-3 to which is slightly higher than that of composite protein-SF/SF-9.52% to a capillary mass is formed by a capillary mass of m-SF-96% and the capillary mass of 2, which is gradually reduced by capillary mass of capillary blood is achieved at a maximum value of 5 to achieve a capillary absorption rate of 96% to 2 to physiological saline after the capillary blood absorption rate of 5 (F-96) is achieved under the same conditions, shows long-acting hemostatic potential.

Test example 6

Based on example 1, the in vitro coagulation performance of the multifunctional medical composite sponges (samples 1 to 3, 5 and comparative example 1) was tested. To sample 1, 0.2mL of fresh anticoagulated whole blood was added dropwise, 0.02mL of CaCl ₂ solution (0.1 mol/L), incubated for 5min, 25mL of deionized water was added, and after shaking for 5min, the absorbance at 545nm was measured by a spectrophotometer and was designated as A _{Sample of} , the absorbance measured by adding 0.2mL of fresh anticoagulated whole blood to 25mL of deionized water was designated as A blank, and the BCI of m-SF/WK/HA was calculated, and the result was shown in FIG. 5, BCI=A _{Sample of} /A _{Blank space} ×100%. As can be seen from fig. 5 (a) and 5 (B), sample 1 was red after being added dropwise to whole blood, had coagulated blood clots on the surface, had no apparent red blood cells around it dissolved out after immersing in deionized water, and had red blood cells around it dissolved out after immersing in deionized water in comparative example 1. As can be seen from FIG. 5 (C), at 1min, sample 1 had a BCI of 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 ability. On one hand, due to the double hydrophilic capability of HA and WK and the stable hierarchical pore structure of m-SF/WK/HA, the liquid absorption speed is improved, the blood viscosity is promoted, and the extrinsic coagulation capability is improved. On the other hand, the composite protein microspheres loaded in the m-SF/WK/HA promote the adhesion and aggregation of platelets and accelerate the endogenous blood coagulation so as to achieve the aim of multiple hemostasis. As the WK content in the m-SF/WK/HA increases, the BCI value slightly decreases, indicating a slight improvement in hemostatic properties of the m-SF/WK/HA, which is related to an improvement in pore structure and liquid absorption capacity. As can be seen from comparing the BCI of sample 1 and sample 5, the addition of the composite protein microspheres advantageously reduces the BCI of m-SF/WK/HA, which is related to the release of calcium ascorbate and protamine from the composite protein microspheres. Ca ²⁺ in calcium ascorbate can be combined with platelet receptors to promote the adhesion and aggregation of platelets, and is beneficial to starting an endogenous hemostasis mechanism. And the calcium ascorbate has the functions of resisting oxidization and inflammation, is favorable for synthesis of collagen and promotes wound healing. The protamine surface in the composite protein microsphere is positively charged, which is beneficial to promoting the aggregation of red blood cells and plays a role in accelerating blood coagulation. Compared with the traditional silk fibroin/hyaluronic acid composite scaffold, the m-SF/WK/HA multifunctional medical composite sponge is beneficial to neutralizing acidic substances of a wound surface on the basis of double hydrophilic capacity of HA and WK, and the microenvironment of the wound surface is changed. In combination, the m-SF/WK/HA HAs the capability of stopping bleeding, protecting wound, and promoting wound healing.

Test example 7

The multifunctional medical composite sponge after blood suction (sample 1 and comparative example 1) was placed on cellulose weighing paper, and the adhesion ability of the material was visually observed after tilting at 60 ° for 30s, and the result is shown in fig. 6. As can be seen from FIG. 6, both sample 1 and comparative example 1 were able to adhere to the substrate initially (A). After 30s, compared with comparative example 1 after blood suction, the sample 1 can stay on the paper surface (B), which shows that the m-SF/WK/HA HAs certain adhesion capability, and is beneficial to better playing the role of blood suction and hemostasis when staying on the wound surface.

Test example 8

Based on example 1, the antibacterial ability of the multifunctional medical composite sponges (sample 1 to sample 3 and comparative example 1) was tested using the shaking method. Weighing 0.5g of multifunctional medical composite sponge, shearing, and sterilizing by ultraviolet rays for later use. Diluting staphylococcus aureus bacterial liquid to 10 ⁵ CFU/mL by using sterile PBS solution, adding 50mL of diluted bacterial liquid into a sample, carrying out shaking culture in a constant-temperature oscillator at 37 ℃ for 1 day, 3 days and 5 days, uniformly coating 100 mu L of bacterial liquid on an LB solid medium, culturing in a constant-temperature incubator at 37 ℃ for 24 hours, counting the number of bacterial colonies, and calculating the bacteriostasis rate. The antibacterial ratio= (1-experimental bacterial liquid concentration/blank bacterial liquid concentration) ×100% and the result is shown in fig. 7. As can be seen from fig. 7, after 1d, the antibacterial rate of sample 1 against staphylococcus aureus was 77.34±4.31%, indicating that the loaded silk fibroin microsphere released the antibacterial agent outwards. As the loading of the composite protein microsphere increases, the antibacterial capacity of m-SF/WK/HA is improved (sample 3 reaches 96.42+/-5.21%), and comparative example 1 HAs no obvious antibacterial capacity. According to the statistical results of the antibacterial rate of different days, the m-SF/WK/HA loaded composite protein microspheres can release the antibacterial agent for a long time, and the released protamine can inhibit the adhesive bacteria of the staphylococcus aureus biological film from producing extracellular polysaccharide, so that the antibacterial agent HAs a long-time antibacterial effect, is beneficial to preventing bleeding wound infection and promoting wound repair.

Test example 9

Based on example 1, toxicity of the multifunctional medical composite sponge on L929 mouse fibroblasts was detected with reference to national standard GB/T16886.5-2017, medical device biological evaluation part 5 in vitro cytotoxicity test. After m-SF/WK/HA (sample 1-sample 3 and comparative example 1) was leached with DMEM medium containing 10% fetal bovine serum for 72 hours, the leaching solution was filtered through a microporous filter membrane of 0.22 μm and the cells were cultured. After 24h incubation, the stock broth was discarded, DMEM containing 10% cck-8 was added to the cells and incubated for 2h, and absorbance was measured at 450nm on a microplate reader after shaking. And calculating the cell viability by using a formula, and judging the influence of m-SF/WK/HA on the proliferation of the L929 cells. Cell viability (%) = (OD _tes-OD_pos)/(OD_neg-OD_pos) ×100. Wherein OD _tes represents the absorbance of the test sample, OD _pos represents the absorbance of the positive control group (cytotoxicity reached 100%), and OD _neg represents the absorbance of the negative control group (control group with cell culture solution only), and the results are shown in fig. 8. As can be seen from FIG. 8, the cell viability of each group of samples after 24 hours was over 80%, indicating that m-SF/WK/HA HAs good cell safety.

Test example 10

Based on example 1, the CCK-8 method was used to detect the proliferation activity of L929 cells on m-SF/WK/HA composite medical sponges. L929 cells were planted on m-SF/WK/HA composite medical sponge (sample 1-sample 3 and comparative example 1), the culture medium was discarded after 1d, 3d and 5d, respectively, the medium was discarded after washing with PBS solution, DMEM containing 10% CCK-8 was added for further incubation for 2 hours, absorbance was measured at 450nm on an microplate reader after shaking, and the results are shown in FIG. 9. As can be seen from FIG. 9, the OD value of each group of materials increases with the increase of the culture days, which indicates that each group of m-SF/WK/HA composite medical sponges can support the growth and proliferation of cells. The OD values of the samples 1-3 are not obviously different from the OD value of the comparative example 1, which shows that the composite protein microsphere loaded by the m-SF/WK/HA HAs no obvious influence on the growth of cells, and the m-SF/WK/HA composite medical sponge HAs antibacterial capability and can support cell proliferation and HAs the potential of promoting wound healing.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (9)

1. The preparation method of the multifunctional medical composite sponge is characterized by comprising the following steps of:

s1, adding a first silk fibroin solution and ethanol into a first mixed solution, and washing and freeze-drying to obtain silk fibroin microspheres, wherein the first mixed solution is prepared by dissolving calcium ascorbate and ascorbic acid in water;

s2, adding the silk fibroin microsphere described in the S1 into a protamine solution, stirring, standing, centrifuging and freeze-drying to obtain a composite protein microsphere;

S3, adding a cross-linking agent and the composite protein microsphere in the S2 after the second mixed solution is balanced at a low temperature, performing oscillation cross-linking reaction, and then performing freeze drying to obtain the multifunctional medical composite sponge, wherein 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 is balanced by firstly standing at 0-4 ℃ for 5-10 min, and then stirring at 20-60 r/min for 30S-600S.

2. The method for preparing a multifunctional medical composite sponge according to claim 1, wherein in S1, the concentration of calcium ascorbate in the first mixed solution is 0.1mg/L-10mg/L, and the concentration of ascorbic acid is 0.1g/L-3g/L;

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

the volume ratio of the first mixed solution to the first silk fibroin solution is 10 (1-5).

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

4. The method for preparing the multifunctional medical composite sponge according to claim 1, wherein in the step S3, the concentration of silk fibroin in the second silk fibroin solution is 5mg/mL-30mg/mL, the molecular weight of silk fibroin is 9kDa-12kDa, the concentration of wool keratin in the wool keratin solution is 2mg/mL-8mg/mL, the molecular weight of wool keratin is 8kDa-14kDa, the concentration of sodium hyaluronate in the sodium hyaluronate solution is 5mg/mL-10mg/mL, and the molecular weight of sodium hyaluronate is 1000kDa-2200kDa;

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 microsphere in the second mixed solution is (1-20): 1-20.

5. The method for preparing a multifunctional medical composite sponge according to claim 1, wherein in S3, the crosslinking agent is N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide.

6. The method for preparing a multifunctional medical composite sponge according to claim 1, wherein 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 a multifunctional medical composite sponge according to claim 1, wherein in S3, the oscillation crosslinking reaction is that stirring is carried out for 120S-240S at a speed of 50r/min-70r/min, then oscillating for 30S-60S at a speed of 60n/min-90n/min and an inclination angle of 45 ° -90 ℃, standing for 5S-10S at a temperature of 0 ° -4 ℃, and then oscillating for 10S-30S at a speed of 30n/min-60n/min and an inclination angle of 45 ° -90 ℃, and standing for 3S-5S at a temperature of 0 ° -4 ℃;

The freeze drying is to freeze at-45 ℃ to-35 ℃ for 6h-8h, at-85 ℃ to-75 ℃ for 10h-12h, and then dry for 48h-72h.

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

9. Use of a multifunctional medical composite sponge according to claim 8 for the preparation of hemostatic materials.