CN110812526B

CN110812526B - PRP-chitosan-silk fibroin composite material and preparation method thereof

Info

Publication number: CN110812526B
Application number: CN201911050578.7A
Authority: CN
Inventors: 韩玎玎; 贺曾; 钟锐; 张学俊; 王红
Original assignee: Chinese Academy Of Medical Science Peking Union Medical College Institute Of Blood Transfusion Chengdu China
Current assignee: Chinese Academy Of Medical Science Peking Union Medical College Institute Of Blood Transfusion Chengdu China
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2022-03-15
Anticipated expiration: 2039-10-31
Also published as: CN110812526A

Abstract

The invention belongs to the technical field of biomaterial synthesis, and particularly relates to a novel PRP-chitosan-silk fibroin composite material capable of rapidly stopping bleeding and a preparation method thereof. The composite material contains 2-4% chitosan solution and 3% silk fibroin solution, then human platelet-rich plasma containing various growth factors and fixed platelet concentration is introduced, and the PRP-chitosan-silk fibroin composite material is prepared by a freeze drying method. The components contained in the material are safe and non-irritant, and compared with the prior art, the PRP with fixed platelet concentration is added in the material, and the silk fibroin is added to change the structure of the base material, so that the coagulation speed of whole blood is improved, the bleeding time and the bleeding amount of a wound are reduced, the hemostatic property of the material is improved, and the hemostatic speed, the pain relieving and the antibacterial property of the wound are promoted.

Description

PRP-chitosan-silk fibroin composite material and preparation method thereof

Technical Field

The invention belongs to the technical field of biomaterial synthesis, and particularly relates to a novel PRP-chitosan-silk fibroin sponge dressing capable of rapidly stopping bleeding and a preparation method thereof.

Background

Uncontrolled blood loss is the second leading cause of death after trauma in everyday life. In some serious accidents, patients often suffer from blood coagulation dysfunction caused by a great amount of blood loss, or serious bleeding of the surgical wound surface occurs in the surgical process, so that the whole wound surface is subjected to blood seepage and the bleeding point is not clear. At this time, the conventional hemostatic methods such as direct compression, ligation and electrocautery are difficult to achieve, and an effective and fast hemostatic material is required.

Coagulation refers to the process by which blood changes from a fluid in a fluid state to a gel state in which it cannot flow. The essence is a complex process of gradual formation of stable insoluble fibrin from unstable platelet emboli by plasma, involving platelet adhesion, aggregation, and changes in red blood cells, white blood cells, vasoconstriction and blood viscosity. Chitosan is a natural material with good biocompatibility, and the hemostasis mechanism of the chitosan is that the chitosan carries a certain amount of charges, can promote erythrocyte aggregation and platelet adhesion, promotes blood coagulation, and completes hemostasis, so the chitosan is widely used in wound hemostasis dressings. Hemostatic bandages and hemostatic powder and the like which take chitosan as a matrix have already been approved by the FDA, and the two materials have become necessary first-aid articles for soldiers in the US and the English army respectively. However, a single chitosan material has a not ideal hemostatic effect on a broad bleeding wound surface, and also has certain limitations in mechanical properties and moisture absorption properties, especially for bleeding caused by irregular wound surfaces and composite vascular rupture, so that chitosan needs to be compounded with other materials with excellent functions to form a novel composite material with complementary advantages.

In recent years, a novel blood extract platelet-rich plasma (PRP) rich in high-concentration platelets is gradually used for studies on wound hemostasis and repair. This is because PRP contains high concentration of platelets, 3-5 times of normal blood, and also contains coagulation factors and fibrinogen, and after activation, the high concentration platelets aggregate and release various growth factors to peripheral tissues, and the activated platelets, the released growth factors, the contained coagulation factors and the fibrinogen in PRP play a crucial role in the process of wound hemostasis and repair, respectively or cooperatively. However, there are many ways to prepare PRP and activate PRP, and no unified standard operation is established clinically, resulting in inconsistent curative effects due to differences in platelet concentration or inconsistent degree of platelet activation. PRP is a high cost biological product and should be used efficiently 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 consolidated. In addition, the source of PRP can be divided into autologous and allogeneic PRP, the advantage of allogeneic PRP lies in that can utilize the whole blood preparation of the healthy donor to get, overcome the patient in autologous PRP and can't draw the problem of whole blood because of the self-health reason, allogenic PRP is different from autologous PRP at the same time, it does not need to draw blood and spend certain time to prepare blood into PRP from patient before using, but a therapeutic means with possibility of preparing ready-made products, therefore researchers can combine allogenic PRP with different biomaterials according to the tissue characteristics repaired, utilize the advantage of substrate composition and structure, realize corresponding therapeutic effect.

The silk is one of the first natural fibers utilized by human beings, has good biocompatibility, the silk fibroin in the silk contains more amino acids with amino groups and carboxyl groups, the hydrophilic groups enable the silk fibroin to be breathable and water-absorbing, and in addition, researches show that the whiteness of the silk fibroin has the effect of regulating and controlling the release of growth factors, and the characteristics enable the silk fibroin material to become one of the research hotspots of medical dressings. However, the application range of the single silk fibroin material is limited due to poor stability and unsatisfactory hemostatic effect of the single silk fibroin material, so researchers are required to overcome the defects after the silk fibroin material is compounded with other materials.

Disclosure of Invention

The invention aims to provide a PRP-chitosan-silk fibroin composite material capable of rapidly stopping bleeding aiming at the technical problems. The composite material contains natural antibacterial chitosan component, natural silk fibroin with good hygroscopicity and PRP with fixed platelet concentration, and has the characteristics of improving the hemostasis performance of the dressing, resisting bacteria, relieving pain and having no stimulation to wounds. It is another object of the invention to further standardize PRP treatment by modifying the substrate structure, accelerating the speed of the red blood cell clot, reducing the proportion of PRP embedded in the material, making efficient use of PRP, while using PPP to adjust PC to PRP with a fixed platelet concentration.

It is another object of the present invention to provide a method for preparing the above composite material.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

a PRP-chitosan-silk fibroin composite material, the components for preparing the composite material are all safe and non-irritant natural components; the composite material comprises the following raw materials in percentage by volume: the volume ratio of the chitosan solution with the mass concentration of 2-4% is 22.5-67.5%, the ratio of the silk fibroin solution with the mass concentration of 3% is 22.5-67.5%, the ratio of the PRP solution is 10%, and the sum of the total volume percentage is 100%.

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

The preparation method of the material comprises the following steps:

1) adding chitosan powder with low molecular weight and deacetylation degree of 85% into 2% acetic acid water solution, and stirring until the powder is completely dissolved to obtain chitosan solution for later use. Low molecular weight chitosan powder, with molecular weight of 50000-190000 Da.

2) Preparation of PRP: PRP is prepared from allogeneic, but not autologous, whole blood.

The method comprises the following specific steps: collecting whole blood of healthy donor, first centrifuging slightly (centrifugal force: 670g centrifugation time: 5min) to remove precipitate and collect supernatant, then centrifuging heavily (centrifugal force: 2683g centrifugation time: 5min) to prepare concentrated Platelet (PC), using supernatant separated after heavy centrifugation as platelet plasma (PPP), and adjusting the concentration of PC platelet to be 500 × 10⁹The PRP solution was prepared for use as a PRP solution having a fixed platelet count.

3) Preparation of silk fibroin solution: with 0.5% Na₂CO₃Boiling natural silkworm cocoon, degumming for three times, and adding ternary solvent (CaCl)₂∶H₂O∶C₂H₅OH molar ratio of 1: 8: 2) dissolving degummed silk to form silk fibroin solution, and dialyzing the solution for 3d (at 4 deg.C). After dialysis, all the solution in the dialysis bag is collected and residues are filtered out by gauze, and the filtered all the solution is used as a silk fibroin solution and is stored at 4 ℃ for later use.

4) Mixing the standby chitosan solution and the silk fibroin solution according to a ratio, continuously stirring in the mixing process until the two solutions are uniformly mixed, and then adding the prepared PRP solution for continuous mixing; and then, the mixed solution is degassed by ultrasound (the temperature is 25 ℃, the working frequency is 40000Hz) for 20min, added into a 24-pore plate, immediately put into a refrigerator at the temperature of-70 ℃ for freezing for 12h, and then freeze-dried by a freeze drier (the vacuum degree is less than 100mbar, and the temperature is-55 ℃) for 36h, thus obtaining the PRP-chitosan-silk fibroin composite material.

The positive effects of the invention are as follows:

the invention utilizes natural chitosan with good biocompatibility, adds PRP (human blood extract) with low volume ratio (10%) into the chitosan, adds fibroin protein which is extracted from natural silkworm cocoon and contains hydrophilic groups, and prepares a novel PRP-chitosan-fibroin protein composite material for rapidly stopping bleeding by a freeze drying method.

And (II) the coagulation speed of whole blood is improved, the bleeding time and the bleeding amount of the wound are reduced, the hemostatic property of the material is improved, the hemostatic speed of the wound is promoted, and the analgesic and antibacterial effects are good.

And (III) accelerating the speed of erythrocyte clotting, reducing the proportion of PRP embedded in the material, efficiently utilizing the PRP by changing the structure of the base material, and simultaneously regulating PC to the PRP with fixed platelet concentration by using PPP, thereby further realizing the standardization of PRP treatment.

Fourthly, the hemostatic property of the material is good; the silk fibroin containing hydrophilic groups has the effects of improving the air permeability of the material, and changing the pore structure and the water absorption of the material and accelerating the speed of erythrocyte clotting in blood by adjusting the different adding amounts of silk fibroin solution, so that the hemostatic property of the material is further improved.

Description of the drawings:

FIG. 1a is a scanning electron microscope image of the composite material prepared in comparative example 1;

FIG. 1b is a scanning electron micrograph of the composite prepared in example 1;

FIG. 1c is a scanning electron micrograph of the composite prepared in example 2;

FIG. 1d is a SEM image of the composite material prepared in example 4.

FIG. 2 is a graph showing the results of in vitro whole blood coagulation of various composite materials (note: PRP/CS-SK-1: 0: comparative example 1 material; PRP/CS-SK-1: 1: example 1 material; PRP/CS-SK-1: 3: example 2 material; PRP/CS-SK-3: 1: example 3 material).

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

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

Detailed Description

The present invention will be described in further detail with reference to specific embodiments for making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.

The technology and the characterization means adopted in the embodiment of the invention comprise the steps of observing the porous structure of the composite material by adopting a scanning electron microscope, measuring the porosity and the water absorption of the material by adopting a soaking method, characterizing the coagulation time of the material by adopting an in-vitro whole blood coagulation method, establishing a rat thoracic and abdominal wall superficial vein wound model, and characterizing the hemostasis performance of the composite material by measuring the bleeding amount of a wound and the wound hemostasis time.

As used herein,% indicates its mass concentration, i.e., wt%, unless otherwise specified.

Example 1:

a PRP-chitosan-silk fibroin composite material: the composite material comprises chitosan, silk fibroin and a human-derived PRP component; the composite material comprises the following raw materials in percentage by volume: 45% of chitosan solution, 45% of silk fibroin solution and 10% of PRP solution, wherein the sum of the percentage contents of the total volume is 100%. Wherein the chitosan solution has a mass concentration of 4%, the silk fibroin solution has a mass concentration of 3%, and the PRP solution has a platelet concentration of 500 × 10⁹And (2) per liter. Mixing the raw materials in proportion, and further processing to obtain the PRP-chitosan-silk fibroin composite material

The preparation method of the composite material comprises the following specific steps:

s1: preparation of 4% chitosan solution: preparing 2% (V: V) acetic acid solution by pure water, dissolving chitosan with the solution to prepare 4% chitosan-acetic acid solution, stirring continuously in the whole process, after completely dissolving chitosan powder, ultrasonically shaking for 10min, standing overnight at room temperature, and storing at 4 ℃ after bubbles completely disappear.

S2: preparation of PRP: 400mL of healthy whole blood meeting the national blood donation standard is collected, and concentrated Platelets (PC) are prepared according to a platelet rich plasma method, wherein the quality of the platelets meets the quality requirements of whole blood and component blood (GB 18469-2012). The platelet-rich plasma method refers to the process of lightly centrifuging collected healthy whole blood, removing sediment and collecting supernatant, and then re-centrifuging to obtain PC. Lightly centrifuging at a centrifugal force of 670g for 5 min; the centrifugal force of the heavy centrifugation is 2683g, and the centrifugation time is 5 min.

Taking supernatant after re-centrifugation as Platelet Poor Plasma (PPP), and adjusting PC to platelet concentration of 500 × 10 with PPP according to the result of measurement by full-automatic cell counter⁹The PRP solution was prepared for use as a PRP solution having a fixed platelet count.

S3: preparing a silk fibroin solution:

s3.1, degumming silk: adding 0.5% of Na₂CO₃After the solution is boiled, the silkworm cocoon is mixed according to the mass volume ratio of 1: 50 (g: mL) was charged with 0.5% Na₂CO₃Boiling the solution for 30min, repeating the steps for two times, washing the degummed silk with deionized water for 3 times, and drying the silk in an oven at 37 ℃.

S3.2: extracting silk fibroin: accurately weighing 8g of dried silk, shredding the weighed silk, and gradually adding into 100ml of ternary solvent (CaCl)₂、H₂O and C₂H₅OH molar ratio of 1: 8: 2), and the whole process is carried out under the condition of controlling the temperature to be 75 ℃ until the silk is completely dissolved, and then the silk is cooled to room temperature. The cooled protein solution was poured into a pre-treated dialysis bag (retentate fraction)The molecular weight is 8 KD-14 KD), after dialysis for 3d with deionized water, the whole dialysis process needs to be carried out at 4 ℃. After dialysis, the whole solution in the dialysis bag was collected, and after the residue was removed by filtration with gauze, the whole solution after filtration was collected and stored at 4 ℃.

S3.3: preparation of 3% silk fibroin solution: weighing one dry glass test tube, and recording the weight as m₁Adding 5ml of the silk fibroin solution after dialysis and filtration into a glass test tube, and weighing the total weight of the test tube and the solution as m₂Then placing the silk fibroin solution at 60 +/-1 ℃ for drying for 12h, continuously weighing the silk fibroin solution until the weight is constant, and calculating the concentration of the silk fibroin solution according to the following formula as m 3: c ═ m₃－m₁)×100％/(m₂－m₁) And adjusting the concentration of the extracted silk fibroin solution to 3% with pure water according to the determined concentration of the silk fibroin solution.

S4: mixing a chitosan solution and a silk fibroin solution according to the volume percentage of 45%: mixing at 45%, stirring continuously during mixing process until the two solutions are mixed uniformly, adding PRP solution 10% of the total volume of the composite material into the mixed solution by mixing method, ultrasonically degassing the stock solution for 20min, adding into 24-pore plate, immediately freezing in a refrigerator at-70 deg.C for 12h, and freeze-drying in a freeze-drying apparatus for 36 h. The conditions of ultrasonic degassing are temperature: 25 ℃, operating frequency: 40000 Hz; the freeze-drying condition of the freeze-drying instrument is vacuum degree: < 100mbar, temperature: -55 ℃.

Example 2:

a PRP-chitosan-silk fibroin composite material is prepared by the following steps:

the 4% chitosan solution (the preparation method is the same as that in example 1) and the 3% silk fibroin solution (the preparation method is the same as that in example 1) are mixed according to the volume percentage of 22.5%: 67.5 percent of the mixture is mixed and continuously stirred in the mixing process until the two solutions are uniformly mixed, and PRP solution accounting for 10 percent of the total volume of the composite material is added into the mixed solution by a mixing method. The preparation procedure of the composite material was similar to the S4 procedure in example 1.

Example 3:

a preparation method of a PRP-chitosan-silk fibroin composite material comprises the following steps:

mixing 4% chitosan solution (prepared by the same method as in example 1) and 3% silk fibroin solution (prepared by the same method as in example 1) according to the total volume ratio of the stock solutions: 67.5%: 22.5 percent of the mixture is mixed, and PRP solution accounting for 10 percent of the total volume of the composite material is added into the mixed solution. The preparation procedure of the composite material was similar to the S4 procedure in example 1.

Example 4:

the 4% chitosan solution (prepared by the same method as in example 1) and the 3% silk fibroin solution (prepared by the same method as in example 1) are mixed according to the volume ratio of 45%: mixing at 45%, and adding PRP solution 10% of the total volume of the stock solution into the mixture by mixing method. And (3) ultrasonically degassing the mixed solution for 20min, adding the mixed solution into a 24-hole plate, immediately and quickly freezing the stock solution by using liquid nitrogen, freezing the stock solution in a refrigerator for 3h, and freeze-drying the stock solution by using a freeze dryer for 36h to obtain the freeze-dried powder.

Example 5:

mixing 4% chitosan solution (prepared by the same method as in example 1) and 3% silk fibroin solution (prepared by the same method as in example 1) according to the total volume ratio of the stock solutions: 50%: mixing 50 percent of the solution, stirring the solution continuously in the mixing process until the two solutions are mixed evenly, ultrasonically degassing the mixed solution for 20min, adding the mixed solution into a 24-pore plate, immediately freezing the mixed solution in a refrigerator at the temperature of 70 ℃ below zero for 12h, and freeze-drying the frozen solution in a freeze dryer for 36h to obtain the chitosan-silk fibroin composite material. Soaking chitosan-silk fibroin material in PRP with 10% volume of the mixed solution by a soaking method to ensure that the material fully absorbs the PRP, freezing the material in a refrigerator at-70 ℃ for 12h, and freeze-drying the material in a freeze-drying apparatus for 36h to obtain the chitosan-silk fibroin material.

Comparative example 1: preparation of PRP-chitosan composite material

The preparation method of the raw materials is the same as that of the example 1, only the preparation step and the addition step of the 3 percent silk fibroin solution are canceled, and the rest steps are the same as those of the example 1. Wherein the volume percentage of the 4% chitosan solution to the PRP solution is 90%: 10 percent.

Comparative example 2: preparation of PRP-chitosan composite material

The preparation method of the raw material is the same as that of the example 1, only the preparation step and the addition step of the 3% silk fibroin solution are cancelled, and the rest steps are the same as those of the example 1. Wherein the volume percentage of the 4% chitosan solution to the PRP solution is 50%: 50 percent.

A scanning electron microscope is adopted to observe the porous structure of the material, the porosity and the water absorption of the material are measured by a soaking method, and the coagulation time of the material and the hemostatic property of the rat thoracic and abdominal wall superficial vein wound model characterization material are characterized by an in vitro whole blood coagulation method.

Experiment 1: and (3) porosity determination:

adding a certain volume of absolute ethyl alcohol into a measuring cylinder, wherein the volume is recorded as V1, putting the material in the embodiment 1 with the diameter of 1.2cm and the thickness of 1cm into the measuring cylinder, standing for 5min to ensure that the material is completely soaked by the absolute ethyl alcohol and no obvious bubbles exist on the surface, and the total volume at the moment is recorded as V2; the material was removed and the volume of absolute ethanol remaining in the cylinder was recorded as V3. The porosity P% of the material is calculated according to the following formula: p% (V1-V3)/(V2-V3) × 100%.

Porosity measurements were performed on examples 1 to 3, and comparative example 1, with the specific results shown in table 1:

TABLE 1 porosity determination results for different materials

As can be seen from table 1, the porosity of the material was significantly increased after adding the silk fibroin solution to the material, wherein the porosity of the material of example 1 was the highest.

The porosity measurements were carried out for example 1 and example 4, and the specific results are shown in table 2:

TABLE 2 porosity measurements of materials prepared by different freezing modes

The results show that the liquid nitrogen freezing stock solution can cause the porosity of the composite material to be reduced, namely, the pores are sparsely distributed on the surface of the material.

Experiment 2: and (3) measuring the water absorption expansion rate:

the material was cut to a suitable size such that the weight of the material was around 40mg, the weighed mass was M0, immersed in 0.01M PBS (pH 7.4) at 37 ℃ for 1h, the material was taken out and weighed mass was M1, and the water absorption W% was calculated according to the following formula: w% (m 1-m 0)/m0 × 100%. The water absorption performance of the material is closely related to the hydrophilicity of the stent, and belongs to one of the physical properties of the hemostatic material. When the material is pressed on a wound, the material with strong water absorption can absorb water in blood, so that the blood viscosity is increased, platelet aggregation is promoted, and blood coagulation is realized.

TABLE 3 Water absorption measurement results of various materials

From the results in table 3, it can be seen that the water absorption rate is increased when silk fibroin is added to the material, wherein the higher the volume ratio of the silk fibroin solution is, the higher the water absorption rate of the material is.

TABLE 4 Water absorption measurement results of materials prepared by different freezing modes

Table 4 shows that the liquid nitrogen frozen stock solution causes the water absorption of the material to decrease, which may be caused by the sparse pore distribution of the material prepared by liquid nitrogen freezing, and thus it can be presumed that the composite material prepared by liquid nitrogen fast freezing stock solution has a lower hemostatic property than the composite material prepared by refrigerator freezing stock solution.

Experiment 3: observation of porous structure:

the materials of examples 1 to 3 and comparative example 1 were cut into about 1cm × 1cm, with a thickness of about 5mm and an upward cross section, the materials were adhered to a metal bracket with a conductive double-sided tape, and the bracket structure was observed at an acceleration voltage of 20kV after gold spraying. It can also be seen from FIG. 1 that the pores of the comparative example 1 material without added silk protein appeared to be ordered and uniform, but the pore size distribution was not uniform. After the silk fibroin is added into the material, the pores of the material in the embodiment 2 are not connected, the pore structure is collapsed, the pores can cause the dressing to be fluffy and fragile, but after the volume distribution of the chitosan and the silk fibroin is adjusted, the pores of the material in the embodiment 1 are distributed orderly and have compact structures, the toughness of the dressing with the pore structure is higher, and the dressing is beneficial to physically blocking wounds and promoting hemostasis.

Experiment 4: in vitro whole blood coagulation assay:

5mL of fresh anticoagulated whole blood is extracted, 0.5mL of 0.1M calcium chloride solution is added, and the mixture is fully and uniformly mixed for standby. The material of example 1 was cut into 1.5cm × 1.5cm thick pieces, and then placed in different test tubes, and the temperature was maintained at 37 ℃. Adding 1mL of prepared anticoagulated human whole blood into a test tube to enable the material to fully infiltrate the blood, placing the test tube into a constant-temperature 37 ℃ shaking table for shaking, taking the material out of the constant-temperature shaking table for 1min, 2min, 5min, 10min and 15min respectively, placing the material into a new test tube, adding 10mL of deionized water into the new test tube, keeping the temperature for 5min, washing out uncoagulated erythrocytes in the dressing, finally collecting the washing liquid in the test tube, and measuring the absorbance of the washing liquid (the wavelength is 540nm) by using a spectrophotometer. Figure 2 shows that the absorbance values of the material after addition of silk fibroin were significantly lower than the material without silk fibroin, indicating that the addition of silk fibroin increased the clotting properties of the material, and that the absorbance values of the material of example 1 were the lowest at each time point, indicating that the clotting properties of the material were best and that blood was almost completely coagulated within 15 min. FIG. 3 shows that the absorbance values of the material of example 1 are close to those of the material of comparative example 2, indicating that adding silk fibroin can increase the hemostatic speed of the material, since chitosan is added to silk fibroin in a volume ratio of 1; 1 and reducing the use of PRP to 10% of the stock solution the whole blood clotting rate of the prepared material was compared to PRP and chitosan 1: 1, the material prepared by mixing is similar, which shows that the structure of the base material can be changed by adjusting the mixing ratio of the chitosan and the silk fibroin, and the hemostatic performance of the material is not influenced under the condition of reducing the use amount of PRP. Fig. 4 shows that the absorbance values of the material of example 1 are significantly lower than those of the material of example 5 in the early and middle stages, which illustrates that the whole blood of the material of example 1 prepared by the mixing method has a faster coagulation speed and exhibits more excellent rapid hemostatic ability, probably because the composite material prepared by the soaking method undergoes two times of freeze drying, and the low temperature freezing may affect the structure of the substrate, and the PRP solution directly permeates into the pores of the substrate by soaking and is then embedded in the material by freeze drying, and this method may affect the binding mode of the platelets in the PRP and the material, thereby affecting the hemostatic ability of the material.

Experiment 5: in vivo hemostasis assay in rats:

15 SD rats were randomly divided into 5 groups of 3 rats each, and the weight of the rats was controlled to 220 g-250 g. After the rats were fixed on the operating table in the supine position, 2% sodium pentobarbital (50mg/kg) was intraperitoneally injected. After the rats are anesthetized, a 3cm transverse incision is made at the middle part of the abdomen, superficial veins of the thoracic abdominal wall are exposed and cut off, the weighed material (weight is recorded as m1) in example 1 is covered on the wound after free bleeding for 3s, the pressure is lightly pressed, timing is started, 1 wound bleeding condition is observed every 10s in the first 30s, 1 wound appearance condition is observed every 5 s after the last 30s, the hemostasis time is recorded, the weight of the material (weight is recorded as m2) is accurately weighed after the wound stops bleeding, and the blood volume is calculated, wherein the bleeding volume is m 1-m 2. From the results in table 5, it can be seen that the hemostatic time of the material is shortened and the blood loss is reduced after adding the silk fibroin, which is substantially the same as the test result of the whole blood coagulation time in vitro, wherein the material of example 1 is the shortest in hemostatic time, only needs 48s and has the least blood loss. As can be seen from the results in table 6, the hemostatic time and blood loss of the material of example 1 are similar to those of the material of comparative example 2, and further illustrate that the ratio of chitosan to silk fibroin is adjusted to 1: 1, the use amount of PRP can be reduced without influencing the hemostatic performance of the material. As is clear from the results in Table 7, the material of example 1 exhibited a shorter hemostatic time than that of example 5 and a smaller amount of blood loss than that of example 5, indicating that the composite material prepared by the mixing method exhibited better hemostatic properties than the composite material prepared by the soaking method

TABLE 5 determination of hemostasis time and blood loss for different dressings

TABLE 6 determination of hemostasis time and blood loss for different dressings

TABLE 7 determination of hemostasis time and blood loss for different dressings

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A preparation method of a PRP-chitosan-silk fibroin composite material is characterized by comprising the following steps:

1) preparation of chitosan solution: adding chitosan powder with low molecular weight and deacetylation degree of 85% into 2% acetic acid water solution, and stirring until the powder is completely dissolved to obtain chitosan solution for later use;

2) preparation of PRP solution: collecting whole blood of healthy donor, preparing PC by rich plasma method, re-centrifuging to obtain supernatant as PPP, and diluting PC to fixed concentration of 500 × 10 with the separated PPP⁹counts/L as PRP solution with fixed platelet count;

3) preparation of silk fibroin solution: using 0.5% of Na₂CO₃Boiling natural silkworm cocoon for removingRepeatedly degumming the silk for three times, dissolving the degummed silk with a ternary solvent to form a silk fibroin solution, dialyzing the solution for 3d, collecting all the solution in a dialysis bag after dialysis is finished, filtering out residues with gauze, taking all the filtered solution as the silk fibroin solution, and storing at 4 ℃ for later use;

4) mixing chitosan solution and silk fibroin solution according to a ratio, continuously stirring in the mixing process until the two solutions are uniformly mixed, adding PRP solution, uniformly mixing, ultrasonically degassing the obtained solution for 20min, adding the obtained solution into a 24-pore plate, immediately freezing the obtained solution in a refrigerator at the temperature of-70 ℃ for 12h, and freeze-drying the frozen solution in a freeze-dryer for 36h to obtain the composite material; the composite material comprises the following raw materials in percentage by volume: 22.5-67.5% of chitosan solution, 22.5-67.5% of silk fibroin solution and 10% of PRP solution, wherein the total volume percentage sum is 100%.

2. The method for preparing the PRP-chitosan-silk fibroin composite material of claim 1, wherein: the material is used for stopping bleeding, improves the coagulation speed of whole blood, reduces the bleeding time and the bleeding amount of wounds, improves the hemostatic property of the material, promotes the hemostatic speed of the wounds, and has good analgesic and antibacterial effects; the molecular weight of the low molecular weight chitosan powder is 50000-190000 Da.

3. The preparation method of the PRP-chitosan-silk fibroin composite material as claimed in claim 1, characterized in that the composite material comprises the following raw materials by volume percentage: 45% of chitosan solution, 45% of silk fibroin solution and 10% of PRP solution, wherein the sum of the percentage contents of the total volume is 100%.

4. The method for preparing the PRP-chitosan-silk fibroin composite material of claim 1, wherein: the mass concentration of the chitosan solution is 2-4%; the mass concentration of the silk fibroin solution is 3%.

5. The method for preparing the PRP-chitosan-silk fibroin composite material of claim 1, wherein: the ternary solutionThe agent is CaCl₂、H₂O and C₂H₅Mixture of OH, CaCl thereof₂、H₂O and C₂H₅The molar ratio of OH is 1: 8: 2.

6. The method for preparing the PRP-chitosan-silk fibroin composite material of claim 1, wherein: the rich plasma method is to firstly carry out light centrifugation on the collected healthy whole blood, remove the sediment and collect the supernatant, and then carry out heavy centrifugation to prepare the PC.

7. The method for preparing PRP-chitosan-silk fibroin composite material as claimed in claim 6, wherein: the centrifugal force of the light centrifugation is 670g, and the centrifugation time is 5 min; the centrifugal force of the heavy centrifugation is 2683g, and the centrifugation time is 5 min.