CN107823694B - A kind of moisturizing double-layer sponge dressing and preparation method thereof - Google Patents

Abstract

The present invention provides a composition, which is composed of silk fibroin (SF), glucose (Glu) and glycerol, and the composition is preferably used for the preparation of wound dressings. The prepared moisturizing sponge dressing can prepare a composite sponge dressing without using a cross-linking agent, which can not only support cell growth but also maintain the micro-humid environment required for wound healing, so as to improve the water absorption rate and low moisturizing rate of the film type dressing. Shortcomings.

Description

A kind of moisturizing double-layer sponge dressing and preparation method thereof

technical field

The invention relates to the technical field of biological tissue engineering and biological materials, in particular to a preparation method of a moisturizing double-layer sponge dressing.

Background technique

The skin is the largest organ of the human body and the first barrier against external stimuli. Skin lesions caused by wounds, abrasions, skin ulcers, and burns can easily lead to bacterial infection, fluid loss, and various complications. Serious threat to human life and health. It is usually protected with wound dressings to prevent wound infection and dehydration, and to provide a moist healing environment for wound repair.

The wound healing process is a complex process, and different wounds and different stages of the same wound have different requirements for dressings. No single material can meet the complex needs of the wound healing process. Existing wound dressings, a single material cannot meet the complex needs of the wound healing process, and the mixing of macromolecular composite materials requires the participation of cross-linking agents. Glutaraldehyde, itself a volatile toxic substance, will cause certain harm to the human body during and after the preparation. Secondly, the dressing is of film type. Compared with sponge type dressings, it lacks interconnected porous structures, which on the one hand leads to unsatisfactory water absorption and air permeability, and on the other hand affects the adhesion and proliferation of its cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a moisturizing sponge dressing and a preparation method thereof. The compound sponge dressing can be prepared without using a cross-linking agent, which can not only support cell growth but also maintain the micro-humid environment required for wound healing, so as to improve the Disadvantages of low water absorption and moisturizing rate of film-type dressings.

A composition is composed of silk fibroin (SF), glucose (Glu) and glycerol. The composition is preferably used in the preparation of wound dressings.

A moisturizing sponge dressing has a porous structure with interconnected pores, and the pore diameter of the pores is 100-300 μm; the dressing comprises the above composition.

The content of silk fibroin in the above dressing is 3-8%, the content of glucose is less than 20% by weight of silk fibroin, and the content of glycerin is less than 1% of the total volume. Preferably, when the glycerol content is 0.5% and the glucose content is 5-10%, the composite material has good water absorption and water retention.

The above dressing is freeze-dried to form a film. Compared with the film type dressing, the dressing has loose and porous, interconnected and uniform pore structure, which is beneficial to the absorption of wound exudate, and at the same time, it is also beneficial to improve the water vapor transmission rate and the growth and proliferation of cells. Specifically, the freezing film formation is pre-freezing at -20° C. for 6-8 hours, placing at -80° C. for 10-24 hours, and freeze-drying for 48-54 hours to form a film.

The present invention forms flexible yet strong, stable porous materials. If the formulation and process of the present invention are not adopted, the problem that the dressing is brittle after molding, or the material is too soft and dense, and the hole structure is not easily formed. Furthermore, phase separation occurs without the use of a cross-linking agent (eg, glutaraldehyde).

Above-mentioned moisturizing sponge dressing, its preparation method comprises the following steps:

(1) Preparation of regenerated silk fibroin solution: Take high-quality silkworm cocoons and cut them into spiral cocoon strips with a width of about 2 mm, boil them in a Na ₂ CO ₃ solution with a concentration of 0.5% at a liquor ratio of 1:50 for 30 min, and repeat the degumming 3 times. Tested with picric acid carmine to obtain completely degummed silk fibroin fibers, which were naturally dried. The completely degummed silk fibroin was added to the ternary solvent (CaCl ₂ : CH ₃ CH ₂ OH : H ₂ O = 1: 2: 8) at a liquor ratio of 1: 10, and placed in a water bath at a temperature of 75±2°C It is completely dissolved to obtain a regenerated silk fibroin solution. The solution was dialyzed with distilled water at regular intervals (times/3h) for 3 days; the dialysate was centrifuged at 4°C and 8000rpm for 10min, filtered and configured to obtain a certain concentration of regenerated silk fibroin. solution concentration);

(2) Preparation of silk fibroin/glucose/glycerin sponge dressing:

Add Glu powder to the silk fibroin solution with a concentration of 4% in turn (25°C, stirring at 300rpm for 10min, constant temperature and low speed to ensure uniformity of the silk fibroin solution, and time span to ensure stirring into a homogeneous sol), 75% Gly solution (25°C, stirring at 300rpm) 40 min), and after defoaming, it was cast into a polyacrylamide petri dish. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film. It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

beneficial effect

1. The present invention prepares a porous sponge dressing with a special structure, with uniform pore size and uniform pore distribution.

2. The hygroscopic sponge dressing obtained by the present invention does not use a cross-linking agent, and still has a uniform texture, is relatively soft, and is loose and porous.

3. The hygroscopic sponge dressing prepared by the present invention has excellent moisture absorption and moisture retention properties, dissolution and loss properties, air permeability and mechanical properties, antibacterial properties and the like.

4. The film of the present invention is relatively soft, thin and transparent, and has the functions of waterproof, breathable and antibacterial.

5. The present invention has a good effect of promoting wound healing, and is beneficial to the growth and migration of cells.

6.SF is a water-insoluble natural fibrin composed of non-polar amino acids (eg: glycine, alanine), which limits the ability to absorb wound exudate to a certain extent. The present invention provides a novel wound dressing, which improves the problem and can absorb excess wound exudate, thereby maintaining the moist environment of the wound.

7. The obtained sponge dressing of the present invention can also be used as a bottom layer, and then a film with waterproof, breathable and antibacterial functions is prepared by using polyurethane as a raw material as a surface layer, and the sponge layer and the film layer are bonded by hot-melt adhesive to obtain a double-layer dressing, better. The dressing is isolated from the external environment, and the obtained material is soft, thin and transparent, and has the functions of waterproof, breathable and antibacterial.

8. The hygroscopic sponge dressing of the present invention is a natural macromolecular biological material with wide sources and low cost, and its shape is easy to mold, breathable and moisturizing, safe and non-toxic, and is suitable for skin wound dressings.

Description of drawings

Figure 1 Forming diagram of sponge dressing

Figure 2 The results of the sponge water absorption experiment

Figure 3 The results of the sponge dissolution rate experiment

Fig. 4 The result of the experiment of sponge water retention rate

Fig. 5 The experimental result of water vapor transmission rate of sponge

Figure 6. Results of sponge mechanics experiment

Figure 7 Results of antibacterial properties of sponges

Fig. 8 Scanning electron microscope results of sponge (SF(A), SF-Glu0%-Gly0.5%(B), SF-Glu5%-Gly0.5%(C), SF-Glu10%-Gly0.5%( D), SF-Glu15%-Gly0.5% (E), SF-Glu20%-Gly0.5% (F))

The water absorption result of Fig. 9 embodiment 7;

The water retention result of Fig. 10 embodiment 7;

Figure 11 The dissolution and loss results of Example 7;

The water vapor transmission rate results of Figure 12 Example 7;

Figure 13 Mechanical results of Example 7;

Bacteriostasis results of Figure 14 Example 7;

Figure 15 Physical picture of double-layer dressing

Figure 16 is a mouse experiment observation diagram of Example 6;

Fig. 17 Wound healing rate of Example 6;

Figure 18 Wound shrinkage rate of Example 6;

Detailed ways

The method of the present invention will be described in detail below with reference to the examples, and the following description is for the purpose of further illustrating the present invention and does not constitute a limitation of the present invention.

Example 1

A preparation method of moisturizing sponge dressing, according to the following steps:

(1) Preparation of regenerated silk fibroin solution:

Take high-quality silkworm cocoons and cut them into spirals, degumming and drying, dialysis, centrifugation and filtration to obtain regenerated silk fibroin with a certain concentration, which is reserved at 4°C;

(2) Preparation of silk fibroin/glucose/glycerin sponge dressing:

Add a certain mass ratio of Glu powder (0%, 5%, 10%, 15%, 20%) to the silk fibroin solution, stir at 25°C, 300rpm for 10min; stir at 25°C, 300rpm for 40min; after defoaming, cast to Polyacrylamide petri dishes. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film (tabletop freeze dryer Thermo, Modulyo-d model). It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

Description of appearance and morphology of this sponge dressing: uniform texture, brittle material, poor softness, and inconspicuous pore structure.

Performance check:

(1) Water absorption performance test

According to the method commonly used in the world to test the water absorption performance of wound dressings, that is, the method formulated by the British Pharmacopoeia for medical dressings: cut the material into a size of 2cm × 2cm, equilibrate at 25 ° C and 65% humidity for 24 hours, and record the weight as W; To simulate wound exudate liquid A (142 mmol NaCl, 2.5 mmol CaCl ₂ ), the material was placed in liquid A of 40 times its own weight, incubated at 37 °C for 24 h, and the wet weight of the material was measured after hanging for 30 s, which was recorded as Wt . The calculation is as follows:

Water absorption rate/%=(Wt-W)/W×100% (1)

The results showed that the addition of Glu significantly increased the water absorption properties of the sponge dressing, and when the addition amount was 15%, the water absorption amount was up to 11.8 times the volume of the sponge itself.

(2) Dissolution property testing:

Take 2cm × 2cm material, equilibrate at 25°C and 65% humidity for 24h, and record the weight as Wa; place the material in a weighing bottle containing 100 times its own weight of liquid A, shake and culture at 37°C for 24h, take out the sponge After drying at 37°C and 50% humidity, equilibrated again for 24h, and weighed as Wb. The calculation is as follows:

Hot water dissolution rate/%=(Wa-Wb)/Wa×100% (2)

The results show that the addition of low amounts (5%, 10%) of Glu can effectively reduce the dissolution-loss of SF sponge, and the dissolution-loss rate is 0, but if Glu is continued to be added, when it exceeds 15%, the dissolution-loss rate increases sharply to 9.26%.

Example 2

A preparation method of moisturizing sponge dressing, according to the following steps:

(1) Preparation of regenerated silk fibroin solution:

Take high-quality silkworm cocoons and cut them into spirals, degumming and drying, dialysis, centrifugation and filtration to obtain regenerated silk fibroin with a certain concentration, which is reserved at 4°C;

(2) Preparation of silk fibroin/glucose/glycerin sponge dressing:

Add a certain mass ratio of Glu powder (0%, 5%, 10%, 15%, 20%) to the silk fibroin solution, stir at 25°C, 300rpm for 10min; then add 0.25% glycerol by volume (pre-prepared 75 % glycerol), stirred at 25° C., 300 rpm for 40 min; after defoaming, it was cast into a polyacrylamide petri dish. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film. It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

Description of appearance and morphology of this sponge dressing: uniform texture, general softness, loose and porous.

Performance detection (detection method is with embodiment 1), and detection result is as follows:

(1) Results of water absorption performance: when the Glu content is 5%, the water absorption is the highest, reaching 12.4 times the volume of the sponge itself; if Glu is continued to be added, the water absorption shows a decreasing trend. When the Glu content is 20%, the water absorption is 20% of its own volume. 10 times.

(2) Dissolution loss performance results: when the amount of Glu added is 0%-10%, the dissolution loss rate is basically zero; but when Glu exceeds 10%, the dissolution loss rate slightly increases to 3.9%.

Example 3

A preparation method of moisturizing sponge dressing, according to the following steps:

(1) Preparation of regenerated silk fibroin solution:

Take high-quality silkworm cocoons and cut them into spirals, degumming and drying, dialysis, centrifugation and filtration to obtain regenerated silk fibroin with a certain concentration, which is reserved at 4°C;

(2) Preparation of silk fibroin/glucose/glycerol sponge dressing:

Add a certain mass ratio of Glu powder (0%, 5%, 10%, 15%, 20%) to the silk fibroin solution, stir at 25°C, 300rpm for 10min; then add 0.5% glycerol by volume (pre-prepared 75 % glycerol), stirred at 25° C., 300 rpm for 40 min; after defoaming, it was cast into a polyacrylamide petri dish. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film. It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

Description of appearance and morphology of this sponge dressing: uniform texture, good material softness, loose and porous.

Performance detection (detection method is with embodiment 1), and detection result is as follows:

(1) Results of water absorption performance: the addition of less than 15% Glu can increase the water absorption rate of the sponge. When the Glu content is 5%, the water absorption capacity is up to 12.5 times its own volume. When the Glu content is 20%, the water absorption is less than 10 times its own volume.

(2) Dissolution performance results: with the increase of Glu content, the dissolution loss rate gradually increased, and when the Glu content was 20%, the highest was 7.5%.

Example 4

A preparation method of moisturizing sponge dressing, according to the following steps:

(1) Preparation of regenerated silk fibroin solution:

Take high-quality silkworm cocoons and cut them into spirals, degumming and drying, dialysis, centrifugation and filtration to obtain regenerated silk fibroin with a certain concentration, which is reserved at 4°C;

(2) Preparation of silk fibroin/glucose/glycerin sponge dressing:

Add a certain mass ratio of Glu powder (0%, 5%, 10%, 15%, 20%) to the silk fibroin solution, stir at 25°C, 300rpm for 10min; then add 0.75% glycerol by volume (pre-prepared 75 % glycerol), stirred at 25° C., 300 rpm for 40 min; after defoaming, it was cast into a polyacrylamide petri dish. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film. It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

Description of appearance and morphology of this sponge dressing: uniform texture, good material softness, loose and porous.

Performance detection (detection method is with embodiment 1), and detection result is as follows:

(1) Water absorption performance test: The addition of Glu reduces the water absorption rate of the sponge. When the Glu content is 20%, the sponge can only absorb about 10 times its own volume of liquid.

(2) Detection of dissolution-loss performance: The dissolution-loss rate increases with the increase of Glu content. When the Glu content is 20%, the dissolution-loss rate is up to 12%.

Example 5

A preparation method of moisturizing sponge dressing, according to the following steps:

(1) Preparation of regenerated silk fibroin solution:

Take high-quality silkworm cocoons and cut them into spirals, degumming and drying, dialysis, centrifugation and filtration to obtain regenerated silk fibroin with a certain concentration, which is reserved at 4°C;

(2) Preparation of silk fibroin/glucose/glycerol sponge dressing:

Add a certain mass ratio of Glu powder (0%, 5%, 10%, 15%, 20%) to the silk fibroin solution, stir at 25°C, 300rpm for 10min; then add 1% glycerol by volume (pre-prepared 75 % glycerol), stirred at 25° C., 300 rpm for 40 min; after defoaming, it was cast into a polyacrylamide petri dish. Pre-freeze at -20°C for 6h, -80°C overnight, freeze-dry for 48h to form a film. It was then treated with 80% methanol for 30 min, washed with distilled water three times, and dried at room temperature.

Description of appearance and morphology of this sponge dressing: uniform texture, good material softness, loose and porous.

Performance detection (detection method is with embodiment 1), and detection result is as follows:

(1) Water absorption performance test: The addition of Glu has little effect on the water absorption rate of the sponge, and its water absorption is less than 10 times the volume of the sponge itself.

(2) Detection of dissolution and loss properties: the overall dissolution rate increases with the increase of Glu, and when Glu is 20%, the highest is about 12%.

Example 6

A kind of preparation of moisturizing sponge dressing (method and proportion design are the same as embodiment 3)

Performance Testing:

(1) Water retention performance test:

Take 2cm×2 size material and incubate at 37°C for 24h, the wet weight is recorded as W ₀ . The water-saturated material was transferred to a dry weighing bottle, placed in a 37°C, 35% humidity incubator for a water loss test, and weighed every 1 h (accumulation time 24 h), recorded as W ₂ /t. Then the water retention rate per unit weight at time t is calculated as follows:

Water retention rate/%=(W ₀ -W ₂ /t)/W ₀ ×100% (3)

The results show that: adding different concentrations of Glu, the final water retention time has no significant difference, the time can be as long as 7h, and the water retention rate is 10%. At 4h, the sponge added with Glu had a higher water retention rate of about 40%.

(2) Moisture permeability test:

Select a suitable cylindrical container to fill 10mL of distilled water, and the cross-sectional area of the container mouth should be smaller than the area of the dressing. The dressing with area A was covered on the container, the mouth of the cup was sealed with parafilm, the whole device was placed in a 37°C, 35% relative humidity environment, and the weight of the whole device was measured regularly. The calculation is as follows:

Water vapor transmission rate/%=Slope×24/A g/m ² /day (4)

In the calculation formula, Slope is the slope of the mass change of the device with time; A is the air-permeable area of the sample.

The results showed that the water vapor transmission rate of the sponges with Glu5% and Glu10% was increased by about 1.6 times to 1600 g/m ² /day compared with the sponge without Glu. When the Glu content exceeds 10%, the water vapor transmission rate shows a downward trend.

(3) Testing of mechanical properties:

The material was cut into a size of 3cm×1cm, equilibrated at 25°C and 65% humidity for 24h, and its thickness was measured. The breaking strength and elongation at break of the material were measured on a universal chemical testing machine at a tensile rate of 10 mm/min.

The results show that when the Glu content is 5%, the maximum breaking strength is 0.45MPa. Continuing to add Glu, its breaking strength showed a downward trend. The addition of 0.5% of Gly significantly increases the elongation at break of the sponge, but if 5% of Glu is added, the elongation at break of the sponge is greatly reduced. Continuing to add Glu can increase the elongation at break of the sponge.

(4) Antibacterial performance testing:

Take the prepared sponge, sterilize it with ultraviolet light, use a PBS solution in a water bath at 37°C for 24 hours, and filter it with a 0.22 μm filter to obtain an extract, which is placed in a 4°C refrigerator for later use. The extracts were co-cultured with Escherichia coli and Staphylococcus aureus for 12 hours, and after dilution, they were spotted on LB agar plates for 12 hours, and the number of colonies was recorded. The bacteriostatic rate was calculated as follows:

Bacteriostatic rate/%=(A-B)/A×100% (5)

In the calculation formula, A is the average colony count of the control group; B is the average colony count of the sample.

The results showed that the bacteriostatic rate of Escherichia coli increased with the increase of the concentration of the extract. The addition of low content of Glu did not hinder the antibacterial activity of the sponge against Escherichia coli, but increased the bacteriostatic rate. Materials, when the concentration of the extract is 0.8%, the sponge has the ability to resist Staphylococcus aureus, and when Glu is added, the low concentration of the extract can also show a certain antibacterial activity. Overall, sponges were less effective against S. aureus than E. coli.

(5) Scanning electron microscope detection:

The samples were sprayed with gold twice by SBC-12 ion sputtering machine, each time for 50s, and placed in JEOL, JSM-6610 scanning electron microscope under low-voltage environment scanning and shooting, voltage 20kV, magnification is shown in the note.

The results showed that the pure SF sponge was lamellar and curled, with low porosity, poor connectivity, and no typical pore structure. When Gly is added, compared with pure SF material, the lamellar structure disappears, and holes begin to appear. Then Glu was gradually added, and an obvious porous structure was formed inside the material. The shape of the pores was relatively regular, the thickness of the pore walls was uniform, and the pore size was about 100-200 μm.

Example 7

A double-layer dressing, the sponge dressing obtained in Example 3 is used as the bottom layer, and the polyurethane film is used as the surface layer, and the sponge layer and the film layer are bonded by hot melt adhesive to obtain a double-layer dressing.

(1) Preparation of polyurethane film: Polyurethane films with different concentrations (2%-20%) were prepared with polyurethane as the solute and N-N-dimethylformamide (DMF) as the solvent. The specific steps are as follows: add a certain amount of polyurethane particles into the DMF solution, mix with a magnetic stirrer at 400 rpm at 60°C for 3 hours, pour into a mold (mold: 5cm×5cm) after defoaming, and dry in an oven at 60°C to form a film.

Experimental results:

1. Morphological observation: when the polyurethane concentration is below 8%, due to the low solute concentration, the film cannot be formed completely and uniformly; when the polyurethane concentration exceeds 12%, the film thickness is thick and the softness is poor, and the function of surface ventilation cannot be achieved. Therefore, the initial screening polyurethane concentration is 8%, 10%, 12%.

2. Water absorption results (Figure 9): when the polyurethane concentration is 8%, and the liquid absorption amount is only 0.4 times its own volume, it has a waterproof effect.

3. Water retention results (Fig. 10): when the polyurethane concentration is 8%, the water retention rate can reach 70%; when the polyurethane concentration is 12%, the water retention rate is 80%. This is because the polyurethane itself has poor water absorption, so it can emit less water.

4. Dissolution loss results (Fig. 11): When the polyurethane concentration is 8%-12%, the dissolution loss rate is lower than 2%.

5. Results of water vapor transmission rate (Fig. 12): When the polyurethane concentration is 8%, the water vapor transmission rate is up to 600g/m2/day.

6. Mechanical results (Fig. 13): when the polyurethane concentration is 8%, the minimum breaking strength is 9 MPa, and the breaking elongation is 380%.

Based on the above screening, when the polyurethane concentration is 8%, the performance is the best, and the following antibacterial experiments are done:

Methods: The polyurethane film was cut to 1×1 cm, placed on LB solid medium, Escherichia coli and Staphylococcus aureus were added to the film and cultured for 24 h, the medium under the film was cut out, the colonies were eluted with PBS and diluted for coating. Observe the colony growth.

Antibacterial results (Fig. 14): The polyurethane film has antibacterial function, and no bacteria grow.

Double-layer composite experiment: the SF-Glu5%-Gly0.5%, SF-Glu10%-Gly0.5% sponge and polyurethane 8% film are composited by hot melt adhesive dot matrix composite technology, the sponge is on the bottom, the film is on the top, and the hot melt The adhesive double layer was compounded, the compounding effect was good, and the sticking was tight, and it was used for animal experiments. The obtained double layer dressing is shown in Figure 15.

Animal experiment:

Balb/c mice (about 25 g body weight) were used to create a full-thickness skin defect wound model. Photographs were taken at 3d, 7d and 11d after injury, and the wound healing rate and wound shrinkage rate were calculated using IPP6.0.

Wound healing rate = (original wound area - residual wound area on day n after injury)/original wound area × 100%

Wound shrinkage rate = wound shrinkage area on the nth day after injury/original wound area × 100%

The results are as follows: Observation map of the mouse experiment (Figure 16)

Wound healing rate (Fig. 17): 3d after injury, the wound healing rate of all dressings was less than 20%, but at 7d, the wound healing was significantly accelerated. Sterile gauze, SF-Glu0%-Gly0.5%, The average wound healing rates of SF-Glu5%-Gly0.5% and SF-Glu10%-Gly0.5% dressings were 35.48%, 18.87%, 83.79% and 44.68%, respectively. On the 11th day, the wound healing rate of the two dressings added with Glu was about 90%.

(2) Wound shrinkage rate (Fig. 18): On the 3rd day after injury, there was little difference in the wound surface shrinkage rate among the groups, but on the 7th day, the shrinkage rate of the SF-Glu5%-Gly0.5% dressing was as high as 80%. Until the 11th day, the wound healing rate of all groups was more than 50%, and it was also the highest level of the two dressing groups added with Glu.

Claims (5)

1. The moisture-retention sponge dressing has a porous structure with interconnected pores, and the pore size of the pores is 100-300 mu m; the dressing comprises a composition consisting of silk fibroin, glucose, glycerol.

2. The sponge dressing of claim 1, wherein the weight of the silk element in the dressing is 3-8%, the glucose content is less than 20% of the weight of the silk element, and the glycerol content is less than 1% of the total volume.

3. The sponge dressing of claim 1 or 2, formed into a film by freeze drying.

4. The sponge dressing as claimed in claim 3, wherein the freeze-dried film is formed by pre-freezing at-20 ℃ for 6-8h, standing at-80 ℃ for 10-24h, and freeze-drying for 48-54 h.

5. The sponge dressing of claim 1 prepared by a process comprising the steps of:

(1) preparing a regenerated fibroin solution: cutting high-quality silkworm cocoon into spiral cocoon strips with width of 2mm, and adding Na with concentration of 0.5% according to bath ratio of 1:50₂CO₃Boiling in the solution for 30min, and repeating degumming for 3 times; detecting with picric acid carmine to obtain completely degummed silk fibroin fiber, and naturally drying and air drying; adding completely degummed fibroin into CaCl at bath ratio of 1:10₂:CH₃CH₂OH:H₂Completely dissolving in ternary solvent O =1:2:8 in water bath at 75 + -2 deg.C to obtain regenerated fibroin solution; dialyzing the solution for 3d by periodically changing distilled water; centrifuging the dialysate at 4 deg.C and 8000rpm for 10min, filtering, and preparing to obtain regenerated fibroin with certain concentration, and storing at 4 deg.C;

(2) preparation of fibroin/glucose/glycerol sponge dressing: adding Glu powder and 75% Gly solution into 4% silk fibroin solution in sequence, defoaming, and casting to a polyacrylamide culture dish; pre-freezing at-20 deg.C for 6 hr, standing overnight at-80 deg.C, and freeze drying for 48 hr to form film; treating with 80% methanol for 30min, washing with distilled water for 3 times, and drying at room temperature.

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