CN110812526A - A kind of PRP-chitosan-silk fibroin composite material and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biomaterial synthesis, in particular to a novel PRP-chitosan-silk fibroin composite material for rapid hemostasis and a preparation method thereof. The composite material contains a chitosan solution with a concentration of 2-4% and a silk fibroin solution with a concentration of 3%, and then introduces human-derived platelet-rich plasma containing a variety of growth factors and a fixed platelet concentration, and is prepared by a freeze-drying method PRP-chitosan-silk fibroin composite material. The ingredients contained in the material of the present invention are safe and non-irritating. Compared with the prior art, the material has increased the coagulation speed of whole blood due to the addition of PRP containing a fixed platelet concentration and the addition of silk fibroin to change the structure of the substrate. Reduce the bleeding time and volume of the wound, improve the hemostasis of the material, and promote the hemostasis speed, pain relief and antibacterial of the wound.

Description

A kind of PRP-chitosan-silk fibroin composite material and preparation method thereof

technical field

The invention belongs to the technical field of biomaterial synthesis, in particular to a novel PRP-chitosan-silk fibroin sponge dressing for rapid hemostasis and a preparation method thereof.

Background technique

Uncontrolled blood loss is the second most common cause of death in everyday life after trauma. In some major accidents, patients are often accompanied by coagulation dysfunction caused by massive blood loss, or severe surgical wound hemorrhage during the operation, resulting in bleeding from the entire wound site and unclear bleeding points. At this time, commonly used hemostasis methods such as direct compression, ligation and electrocautery are difficult to achieve, and materials that can effectively and quickly stop bleeding are required.

Coagulation is the process by which blood changes from a fluid state to an immobile gel state. Its essence is a complex process in which plasma gradually forms stable insoluble fibrin from unstable platelet emboli. The whole process involves platelet adhesion and aggregation, as well as changes in red blood cells, white blood cells, vasoconstriction and blood viscosity. Wait. Chitosan is a natural material with good biocompatibility. Its hemostatic mechanism is that it carries a certain amount of electric charge, which can promote red blood cell aggregation and increase platelet adhesion, promote blood coagulation, and complete hemostasis. Therefore, it is widely used in Hemostatic wound dressings. Hemostatic bandages and hemostatic powders based on chitosan have been approved by the FDA, and the two materials have become necessary first aid items for the US military and British soldiers respectively. However, the hemostatic effect of a single chitosan material is not ideal for extensive bleeding wounds, and it also shows certain limitations in mechanical properties and hygroscopic properties, especially for the bleeding caused by irregular wounds and composite blood vessel rupture. It is necessary to composite chitosan with other materials with excellent functions to form new composite materials with complementary advantages.

In recent years, platelet-rich plasma (PRP), a new blood extract rich in high-concentration platelets, has been gradually used in the research of wound hemostasis and repair. This is because PRP contains a high concentration of platelets, which is 3-5 times that of normal blood, and also contains coagulation factors and fibrinogen. After activation, high-concentration platelets will aggregate and release a variety of growth factors. Peripheral tissue, activated platelets, released growth factors, contained coagulation factors and fibrinogen in PRP all play crucial roles in wound hemostasis and repair, respectively or cooperatively. However, at present, there are many ways to prepare and activate PRP, and there is no unified standardized operation standard in clinical practice, resulting in differences in platelet concentration or inconsistency in platelet activation, resulting in inconsistent efficacy. PRP is a high-cost biological product that should be efficiently utilized during use. If the concentration of platelets in PRP can be fixed during the preparation of PRP, the efficacy of PRP technology can be further unified. In addition, the source of PRP can be divided into autologous and allogeneic. The advantage of allogeneic PRP is that it can be prepared from the whole blood of healthy blood donors, which overcomes the problem that patients in autologous PRP cannot draw whole blood due to their own health reasons. Different from autologous PRP, it does not need to draw blood from the patient before use and spend a certain amount of time to prepare the blood into PRP. It is a treatment method with the possibility of preparing a ready-made product. Therefore, researchers based on the characteristics of the repaired tissue , Allogeneic PRP can be combined with different biological materials, and the corresponding therapeutic effect can be achieved by taking advantage of the composition and structure of the substrate.

Silk is one of the earliest natural fibers used by human beings and has good biocompatibility. The silk fibroin in silk contains many amino acids with amino and carboxyl groups. These hydrophilic groups make silk fibroin breathable, Water absorption, and studies have found that silk fibroin can regulate the release of growth factors, these characteristics make silk fibroin material one of the research hotspots of medical dressings. However, the poor stability and unsatisfactory hemostatic effect of a single silk fibroin material limit its application range. Therefore, researchers need to compound silk fibroin with other materials to overcome these shortcomings.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above-mentioned technical problems, and propose a PRP-chitosan-silk fibroin composite material that can quickly stop bleeding. The composite material contains natural and antibacterial chitosan, natural silk fibroin with good hygroscopicity and PRP with a fixed platelet concentration, which can not only improve the hemostatic properties of the dressing, but also have the characteristics of antibacterial, analgesic and non-irritating to wounds . Another object of the present invention is to further realize the efficient use of PRP by changing the structure of the substrate, accelerating the rate of red blood cell clotting, reducing the proportion of PRP embedded in the material, and using PPP to adjust PC to PRP with a fixed platelet concentration. Standardization of PRP treatment.

Another object of the present invention is to provide a method for preparing the above-mentioned composite material.

In order to achieve the above object, the concrete technical scheme of the present invention is:

A PRP-chitosan-silk fibroin composite material, the components for preparing the composite material are all safe and non-irritating natural components; the composite material includes the following raw materials by volume percentage: The volume ratio of chitosan solution is 22.5-67.5%, the proportion of silk fibroin solution with a mass concentration of 3% is 22.5%-67.5%, the proportion of PRP solution is 10%, and the sum of the total volume percentage is 100% .

Preferably, the chitosan solution with a mass concentration of 2-4% accounts for 45% of the volume, the silk fibroin solution with a mass concentration of 3% accounts for 45%, and the PRP solution accounts for 10%. The sum is 100%.

The preparation method of the above-mentioned material comprises the following steps:

1) Add low molecular weight chitosan powder with a degree of deacetylation of 85% into a 2% acetic acid aqueous solution, and stir until the powder is completely dissolved to obtain a chitosan solution for use. Low molecular weight chitosan powder, its molecular weight is 50000-190000Da.

2) Preparation of PRP: PRP was prepared from allogeneic whole blood instead of autologous whole blood.

The specific steps are: collecting whole blood from healthy blood donors, first lightly centrifuging (centrifugal force: 670g centrifugation time: 5min) to remove the sedimentation and collecting the supernatant, then re-centrifugation (centrifugation force: 2683g centrifugation time: 5min) to prepare platelet concentrate ( plateletconcentrate, PC), and the supernatant separated after recentrifugation was used as plateletpoor plasma (PPP), and the PC platelet concentration was adjusted to a fixed concentration of 500 × ¹⁰⁹ /L with the separated PPP, as a PRP solution for fixing the number of platelets, ready for use.

3) Preparation of silk fibroin solution: after boiling natural silkworm cocoons with 0.5% Na ₂ CO ₃ for three times for degumming, the molar ratio of ternary solvent (CaCl ₂ : H ₂ O : C ₂ H ₅ OH is 1:8:2) ) to dissolve the degummed silk to form a silk fibroin solution, and the solution was dialyzed for 3d (at 4°C). After the dialysis, the whole solution in the dialysis bag was collected and the residues were filtered off with gauze.

4) Mix the standby chitosan solution and the silk fibroin solution according to the proportion, keep stirring during the mixing process until the two solutions are evenly mixed, then add the prepared PRP solution to continue mixing; then the mixed solution is sonicated (Temperature: 25°C, working frequency: 40000Hz) After degassing for 20min, add it to a 24-well plate, immediately put it into a -70°C refrigerator for 12h, and then freeze-dry it in a freeze dryer (vacuum degree: <100mbar, temperature: -55°C) for 36h to obtain PRP-chitosan-silk fibroin composite material.

The positive effects of the present invention are reflected in:

(1), the present invention utilizes natural chitosan with good biocompatibility, adds the human blood extract PRP of low volume ratio (10%) to it, and adds the chitosan extracted from natural silkworm cocoons containing hydrophilic A new type of PRP-chitosan-silk fibroin composite material with rapid hemostasis was prepared by freeze-drying method.

(2) It improves the coagulation speed of whole blood, reduces the bleeding time and volume of the wound, improves the hemostasis of the material, promotes the hemostasis speed of the wound, and has good analgesic and antibacterial effects.

(3) By changing the structure of the substrate, accelerating the rate of red blood cell clotting, reducing the proportion of PRP embedded in the material, using PRP efficiently, and using PPP to adjust PC to PRP with a fixed platelet concentration, further realizing the efficacy of PRP therapy standardization.

(4) The hemostatic property of the material is good; the addition of silk fibroin containing a hydrophilic group, one is to improve the air permeability of the material, and the other is to change the pore structure of the material by adjusting the amount of silk fibroin solution added. It absorbs water and accelerates the clotting of red blood cells in the blood, thereby further improving the hemostasis of the material.

Description of drawings:

Fig. 1a is the scanning electron microscope picture of the composite material prepared in Comparative Example 1;

Fig. 1b is the scanning electron microscope picture of the composite material prepared in Example 1;

Fig. 1c is the scanning electron microscope picture of the composite material prepared in Example 2;

FIG. 1d is a scanning electron microscope picture of the composite material prepared in Example 4. FIG.

Figure 2 is a graph showing the results of in vitro whole blood coagulation of different composite materials (Note: PRP/CS-SK-1:0: material of Comparative Example 1; PRP/CS-SK-1:1: material of Example 1; PRP/CS -SK-1:3: material of example 2; PRP/CS-SK-3:1: material of example 3).

Figure 3 is a graph showing the results of in vitro whole blood coagulation of different composite materials (Note: PRP/CS 1:1: material of Comparative Example 2; PRP/CS-SK-1:1: material of Example 1)

Figure 4 is a graph showing the results of in vitro whole blood coagulation of composite materials prepared by different freezing methods (Note: mixing: material of Example 1; Soak: material of Example 5)

Detailed ways

In order to make the purpose of the invention, technical solutions and advantages of the application more clearly understood, the present invention will be further described in detail below in conjunction with the specific embodiments, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples.

The techniques and characterization methods used in the embodiments of the present invention include observing the porous structure of the composite material by scanning electron microscope, measuring the porosity and water absorption of the material by immersion method, characterizing the coagulation time of the material by in vitro whole blood coagulation method, and establishing the rat chest The superficial abdominal vein wound model was used to characterize the hemostatic properties of the composites by measuring the amount of wound bleeding and wound hemostasis time.

Unless otherwise specified, the % used in this application refers to its mass concentration, that is, wt%.

Example 1:

A PRP-chitosan-silk fibroin composite material: the composite material contains chitosan, silk fibroin and human-derived PRP components; the composite material includes the following raw materials by volume percentage: chitosan solution 45%, silk fibroin solution 45%, PRP solution 10%, the sum of the total volume percentage is 100%. The mass concentration of the chitosan solution is 4%, the mass concentration of the silk fibroin solution is 3%, and the platelet concentration of the PRP solution is 500×10 ⁹ /L. The raw materials are mixed in proportion and further processed to obtain the PRP-chitosan-silk fibroin composite material

The specific preparation steps of the composite material are as follows:

S1: Preparation of 4% chitosan solution: take pure water to prepare 2% (V:V) acetic acid solution, use this solution to dissolve chitosan, and prepare a 4% chitosan-acetic acid solution. The whole process Stir continuously, after the chitosan powder is completely dissolved, ultrasonically vibrate for 10 minutes, and then stand at room temperature overnight, and store at 4°C after the bubbles completely disappear.

S2: Preparation of PRP: Collect 400 mL of healthy whole blood that meets the national blood donation standard, and prepare platelet concentrate (PC) according to the platelet-rich plasma method. The platelet-rich plasma method refers to the healthy whole blood collected by light centrifugation first, and the supernatant is collected by removing the sediment, and then centrifuging again to prepare PC. Light centrifugation, its centrifugal force is 670g, centrifugation time 5min; described heavy centrifugation, its centrifugal force is 2683g, centrifugation time 5min.

The supernatant after recentrifugation was taken as platelet poor plasma (PPP), and the PC was adjusted to a platelet concentration of 500 × 10 ⁹ /L with PPP according to the results of the automatic cell counter. The number of PRP solutions, spare.

S3: Preparation of silk fibroin solution:

S3.1 Silk degumming: after boiling the 0.5% Na ₂ CO ₃ solution, put the cocoons into the 0.5% Na ₂ CO ₃ solution at a mass-volume ratio of 1:50 (g:mL), boil for 30 minutes, repeat twice before using The degummed silk was rinsed three times with deionized water and dried in a 37°C oven.

S3.2: Silk Fibroin Extraction: Accurately weigh 8 g of dried silk, tear the weighed silk into pieces and gradually add it to 100 ml of a ternary solvent (molar ratio of CaCl ₂ , H ₂ O and C ₂ H ₅ OH) 1:8:2), the whole process should be temperature-controlled at 75°C until the silk is completely dissolved and then cooled to room temperature. The cooled protein solution was poured into a pretreated dialysis bag (molecular weight cut-off 8KD-14KD), and after dialysis with deionized water for 3 days, the entire dialysis process should be carried out at 4°C. After dialysis, collect all the solutions in the dialysis bag, filter the residue with gauze, collect all the filtered solutions, and store them at 4°C.

S3.3: Preparation of 3% silk fibroin solution: take a dry glass test tube, weigh its weight and mark it as m ₁ , add 5 ml of dialysis-filtered silk fibroin solution to the glass test tube, and weigh the test tube with The total weight of the solution, denoted as m ₂ , and then baked at 60±1°C for 12 hours, continuously weighed until constant weight, denoted as m 3 , the concentration of silk fibroin solution was calculated according to the following formula: C=(m ₃ -m ₁ )×100%/(m ₂ −m ₁ ), according to the measured concentration of the silk fibroin solution, the concentration of the extracted silk fibroin solution was adjusted to 3% with pure water.

S4: Mix the chitosan solution and the silk fibroin solution according to the volume percentage of 45%: 45%, and keep stirring during the mixing process until the two solutions are evenly mixed. The PRP solution with a volume ratio of 10% was added to a 24-well plate after ultrasonic degassing for 20 minutes, immediately placed in a -70°C refrigerator for 12 hours, and then freeze-dried for 36 hours with a freeze dryer. The conditions of ultrasonic degassing are temperature: 25°C, working frequency: 40000Hz; the conditions for freeze-drying of freeze dryer are vacuum degree: <100mbar, temperature: -55°C.

Example 2:

A PRP-chitosan-silk fibroin composite material, the preparation method of which is as follows:

Mix the 4% chitosan solution (the preparation method is the same as that of Example 1) and the 3% silk fibroin solution (the preparation method is the same as that of Example 1) according to the volume percentage of 22.5%: 67.5%, and keep stirring during the mixing process until The two solutions were uniformly mixed, and a PRP solution accounting for 10% of the total volume of the composite material was added to the mixed solution by a mixing method. The preparation steps of the composite material are similar to the S4 step in Example 1.

Example 3:

The preparation method of a PRP-chitosan-silk fibroin composite material is as follows:

Mix 4% chitosan solution (the preparation method is the same as that of Example 1) and 3% silk fibroin solution (the preparation method is the same as that of Example 1) according to the total volume ratio of the original solution: 67.5%: 22.5%, and add the compound to the mixed solution. The total volume of material is 10% PRP solution. The preparation steps of the composite material are similar to the S4 step in Example 1.

Example 4:

The preparation method of a PRP-chitosan-silk fibroin composite material is as follows:

The 4% chitosan solution (the preparation method is the same as that of Example 1) and the 3% silk fibroin solution (the preparation method is the same as that of Example 1) are mixed according to the volume ratio of 45%: 45%, and the total stock solution is added to the mixed solution by the mixing method. 10% PRP solution by volume. The mixed solution was ultrasonically degassed for 20 minutes and then added to a 24-well plate. The stock solution was immediately frozen with liquid nitrogen, placed in a refrigerator for 3 hours, and then freeze-dried for 36 hours using a freeze dryer.

Example 5:

A PRP-chitosan-silk fibroin composite material, the preparation method of which is as follows:

Mix the 4% chitosan solution (the preparation method is the same as that of Example 1) and the 3% silk fibroin solution (the preparation method is the same as that of Example 1) according to the total volume ratio of the original solution: 50%: 50%. Stir until the two solutions are evenly mixed, add the mixture to a 24-well plate after ultrasonic degassing for 20 min, immediately put it in a -70 °C refrigerator for 12 h, and then freeze-dry it in a freeze dryer for 36 h to obtain chitosan-silk fibroin. composite material. By soaking method, the chitosan-silk fibroin material was soaked in PRP with a volume percentage of 10% of the above mixture, after the material fully absorbed PRP, the material was placed in a -70 °C refrigerator for 12 hours and then freeze-dried. Freeze-dried for 36h.

Comparative Example 1: Preparation of a PRP-Chitosan Composite

The preparation method of the raw materials is the same as that in Example 1, except that the preparation steps and the adding steps of the 3% silk fibroin solution are omitted, and the rest of the steps are the same as those in Example 1. The volume percentage of 4% chitosan solution and PRP solution is 90%: 10%.

Comparative Example 2: Preparation of a PRP-Chitosan Composite

The preparation method of the raw material is the same as that in Example 1, except that the preparation steps and the adding steps of the 3% silk fibroin solution are omitted, and the remaining steps are the same as those in Example 1. The volume percentage of 4% chitosan solution and PRP solution is 50%:50%.

The porous structure of the material was observed by scanning electron microscope, the porosity and water absorption of the material were determined by immersion method, the clotting time of the material was characterized by in vitro whole blood coagulation method, and the hemostasis of the material was characterized by the rat thoracic and abdominal wall superficial vein wound model.

Experiment 1: Porosity determination:

A certain volume of absolute ethanol was added to the graduated cylinder, the volume was denoted as V1, the material in Example 1 with a diameter of 1.2 cm and a thickness of 1 cm was put into it, and allowed to stand for 5 minutes, so that the material was completely saturated with absolute ethanol and the surface was free of If there are obvious bubbles, the total volume at this time is recorded as V2; the material is taken out, and the volume of anhydrous ethanol remaining in the graduated cylinder is recorded as V3. The porosity P% of the material is calculated according to the following formula: P%=(V1-V3)/(V2-V3)×100%.

The porosity of Examples 1 to 3, and Comparative Example 1 were measured, and the specific results are shown in Table 1:

Table 1 Porosity measurement results of different materials

It can be seen from Table 1 that after adding silk fibroin solution to the material, the porosity of the material is significantly increased, and the material of Example 1 has the highest porosity.

The porosity of Example 1 and Example 4 was measured, and the specific results are shown in Table 2:

Table 2 Measurement results of porosity of materials prepared by different freezing methods

The results show that freezing the stock solution in liquid nitrogen will reduce the porosity of the composite material, that is, the pores are sparsely distributed on the surface of the material.

Experiment 2: Determination of water swelling ratio:

Cut the material into a suitable size, so that the weight of the material is about 40mg, weigh the mass as m0, immerse it in 0.01M PBS (pH 7.4) at 37°C for 1 h, take out and weigh the mass, denoted as m1, and the water absorption rate W % is calculated according to the following formula: W%=(m1-m0)/m0×100%. The water absorption property of the material is closely related to the hydrophilicity of the stent, which is one of the physical properties of the hemostatic material. When the material is pressed on the wound, the highly absorbent material will absorb the water in the blood, resulting in an increase in blood viscosity to promote platelet aggregation and blood coagulation.

Table 3 Measurement results of water absorption of different materials

According to the results in Table 3, after adding silk fibroin to the material, the water absorption rate increases, and the higher the volume ratio of silk fibroin solution occupies, the higher the water absorption rate of the material.

Table 4 Measurement results of water absorption of materials prepared by different freezing methods

Table 4 shows that liquid nitrogen freezing stock solution will lead to a decrease in the water absorption of the material, which may be caused by the sparse pore distribution of the material prepared by liquid nitrogen freezing. It can be speculated that the hemostasis of the composite material prepared by liquid nitrogen quick freezing stock solution It is weaker than the composite material prepared from the frozen stock solution in the refrigerator.

Experiment 3: Observation of Porous Structure:

The materials of Examples 1 to 3 and Comparative Example 1 were cut into about 1cm×1cm, about 5mm thick, with the cross section facing up, and the materials were adhered to the metal bracket with conductive double-sided The scaffold structure was observed under accelerating voltage. It can also be seen from Figure 1 that although the pore performance of the material of Comparative Example 1 without adding silk protein is orderly and uniform, the pore size distribution is not uniform. After adding silk fibroin to the material, the pores of the material of Example 2 showed incoherence, and the pore structure collapsed, which would cause the dressing to be fluffy and brittle, but after adjusting the volume of chitosan and silk fibroin, the example 1. The pore distribution of the material becomes orderly and the structure is compact. The toughness of the dressing with such a pore structure will be higher, and at the same time, it will be beneficial to physically block the wound and promote hemostasis.

Experiment 4: In vitro whole blood coagulation assay:

Draw 5 mL of fresh anticoagulated whole blood, add 0.5 mL of 0.1 M calcium chloride solution, and mix well for use. The material in Example 1 was cut into a size of 1.5cm×1.5cm and a thickness of 1cm, then put into different test tubes and kept at 37°C. Take 1 mL of the prepared anticoagulated human whole blood and add it to the test tube to make the material fully infiltrate the blood, put the test tube into the constant temperature 37 ℃ shaker and shake, and the material is taken out from the constant temperature shaker at 1min, 2min, 5min, 10min and 15min respectively. Put into a new test tube, add 10 mL of deionized water to the new test tube and keep it for 5 minutes, wash out the uncoagulated red blood cells in the dressing, and finally collect the washing liquid in the test tube, and measure the absorbance of the washing liquid with a spectrophotometer (wavelength=540nm) . Figure 2 shows that after adding silk fibroin, the absorbance value of the material is significantly lower than that of the material without silk fibroin, indicating that adding silk fibroin will improve the coagulation of the material, and the absorbance value of the material of Example 1 at each time point All were the lowest, indicating that the coagulation of the material was the best, and the blood was almost completely coagulated within 15 minutes. Figure 3 shows that the absorbance value of the material of Example 1 is close to that of the material of Comparative Example 2, indicating that adding silk fibroin can improve the hemostasis rate of the material, because chitosan and silk fibroin are mixed in a volume ratio of 1; 1 and reduce the PRP The whole blood coagulation rate of the prepared material is similar to that of the material prepared by mixing PRP and chitosan 1:1, indicating that the substrate can be changed by adjusting the mixing ratio of chitosan and silk fibroin. The structure of the material does not affect the hemostatic properties of the material under the condition of reducing the amount of PRP used. Figure 4 shows that the absorbance value of the material of Example 1 is significantly lower than that of the material of Example 5 in the early and middle stages, indicating that the material of Example 1 prepared by the mixing method has a faster coagulation speed of whole blood and better rapid hemostasis ability. It may be because the composites prepared by the immersion method have undergone two freeze-drying processes, and the low-temperature freezing may affect the structure of the substrate. At the same time, the PRP solution directly penetrates into the pores of the substrate through immersion, and is then embedded in the material by freeze-drying. This method It may affect how platelets bind to the material in PRP, thereby affecting the hemostatic properties of the material.

Experiment 5: In vivo hemostasis assay in rats:

Fifteen SD rats were randomly divided into 5 groups with 3 rats in each group. The weight of rats was controlled at 220g-250g. After the rats were supine and fixed on the operating table, 2% sodium pentobarbital (50 mg/kg) was intraperitoneally injected. After the rat was anesthetized, a 3cm transverse incision was made in the middle of the abdomen to expose the superficial thoracic and abdominal veins and cut them off. Gently press and start timing at the same time, observe the bleeding of the wound every 10 s for the first 30 s, and observe the appearance of the wound every 5 s after 30 s, and record the hemostasis time. m2), and calculate the amount of bleeding, the amount of bleeding = m1-m2. It can be known from the results in Table 5 that after adding silk fibroin, the hemostasis time of the material is shortened and the blood loss is reduced, which is basically consistent with the results of the in vitro whole blood coagulation time test. least. It can be seen from the results in Table 6 that the hemostasis time and blood loss of the material of Example 1 are similar to those of the material of Comparative Example 2, which further illustrates that by adjusting the mixing ratio of chitosan and silk fibroin to 1:1, the amount of PRP used can be reduced at the same time. It does not affect the hemostatic properties of the material. It can be known from the results in Table 7 that the hemostasis time of the material of Example 1 is shorter than that of Example 5, and the blood loss is also less than that of Example 5, indicating that the hemostasis of the composite material prepared by the mixing method is better than that of the composite material prepared by the soaking method.

Table 5 Measurement results of hemostasis time and blood loss of different dressings

Table 6 Measurement results of hemostasis time and blood loss of different dressings

Table 7 Hemostasis time and blood loss measurement results of different dressings

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (8)

1. A PRP-chitosan-silk fibroin composite material, characterized in that the composite material comprises the following raw materials by volume percentage: chitosan solution 22.5-67.5%, silk fibroin solution 22.5%-67.5% , PRP solution 10%, the sum of the total volume percentage is 100%. 2. The PRP-chitosan-silk fibroin composite material according to claim 1, characterized in that the composite material comprises the following raw materials by volume percentage: 45% chitosan solution, 45% silk fibroin solution , PRP solution 10%, the sum of the total volume percentage is 100%. 3. The PRP-chitosan-silk fibroin composite material according to claim 1 or claim 2, wherein the mass concentration of the chitosan solution is 2-4%; the mass concentration of the silk fibroin solution is 3%. 4. The preparation method of the PRP-chitosan-silk fibroin composite material according to any one of claims 1 to 3, characterized in that it comprises the following steps: 1) Configuration of chitosan solution: add chitosan powder with low molecular weight and a degree of deacetylation of 85% into 2% acetic acid aqueous solution, stir until the powder is completely dissolved, and obtain a chitosan solution for use; 2) Preparation of PRP solution: Collect whole blood of healthy blood donors and prepare PC according to the enriched plasma method. The supernatant liquid separated after re-centrifugation is used as PPP, and the PC is diluted to a fixed concentration of 500× with the separated PPP. 10 ⁹ /L, as a PRP solution with a fixed number of platelets. 3) Preparation of silk fibroin solution: degumming by boiling natural silk cocoons with _0.5 % _Na2CO3 , repeating the degumming three times, dissolving the degummed silk with a ternary solvent to form a silk fibroin solution, dialyzing the solution for 3d, dialyzing After completion, collect all the solutions in the dialysis bag and filter the residues with gauze, and all the filtered solutions are used as silk fibroin solution and stored at 4°C for future use; 4) Mix the chitosan solution and the silk fibroin solution according to the proportion, and keep stirring during the mixing process until the two solutions are evenly mixed, then add the PRP solution and mix evenly, and then add the obtained solution to ultrasonic degassing for 20 minutes. 24-well plate, immediately put into -70 ℃ refrigerator to freeze for 12 hours, and then freeze-dried by freeze dryer for 36 hours to obtain composite materials. 5. The preparation method of PRP-chitosan-silk fibroin composite material according to claim 4, wherein the ternary solvent is a mixture of CaCl ₂ , H ₂ O and C ₂ H ₅ OH. The molar ratio of CaCl ₂ , H ₂ O to C ₂ H ₅ OH was 1:8:2. 6. The preparation method of PRP-chitosan-silk fibroin composite material according to claim 4, characterized in that: the enriched pulp method refers to the healthy whole blood collected by light centrifugation first, and the sedimentation is removed to collect the supernatant. After the solution, re-centrifugation was used to prepare PC. 7. The preparation method of the PRP-chitosan-silk fibroin composite material according to claim 6, characterized in that: the centrifugal force of the light centrifugation is 670 g, and the centrifugal time is 5 min; the centrifugal force of the heavy centrifugation is 2683g, centrifugation time 5min. 8 . The preparation method of the PRP-chitosan-silk fibroin composite material according to claim 4 , wherein the low molecular weight chitosan powder has a molecular weight of 50000-190000 Da. 9 .

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