CN117323461A - Hemostatic material and preparation method thereof - Google Patents

Abstract

The invention discloses a hemostatic material and a preparation method thereof, wherein the hemostatic material comprises lac and gelatin; adding gelatin into lac solution, mixing, and lyophilizing. The hemostatic material has strong hemostatic capability, and compared with medical gauze and commercial gelatin sponge, the hemostatic material obviously shortens hemostatic time and blood loss; the water absorption rate and the water absorption rate are high, and the wound can be quickly plugged after water absorption; the liquid can be absorbed rapidly when contacting the wound, the aggregation of the platelet and the posterior cell is accelerated, and the hemostasis is rapid; the hemostatic patch also has good shape memory performance, can quickly absorb blood for irregular and deep wounds, quickly recover the original state, block the wounds, and has good hemostatic performance and biocompatibility; and has good antibacterial property and coagulation property, and has application prospect in biomedical applications such as wound coagulation hemostasis and tissue repair.

Description

Hemostatic material and preparation method thereof

Technical Field

The invention relates to a medical material and a preparation method thereof, in particular to a medical hemostatic material and a preparation method thereof, and belongs to the technical field of biomedical materials.

Background

Bleeding is a common typical event of traffic accidents, surgery, war, natural disasters, etc. Massive hemorrhage is susceptible to excessive blood loss and death, and it is counted that more than 30% of traumatic deaths worldwide are attributable to massive hemorrhage. Conventionally, gauze and bandages are main hemostatic materials for rapidly controlling bleeding, and in order to enhance hemostatic effects of conventional gauze, a number of substances having a blood coagulation function such as thrombin, protamine, phenolsulfoethylamine, aminocaproic acid, tranexamic acid, and the like have been used to enhance hemostatic effects of conventional gauze. Currently, there are products such as Combat gauze, hemCon bandages, surgicel and the like on the market, and although these products have remarkable hemostatic capabilities for superficial lesions, experimental results indicate that most of the products cannot deal with severe bleeding cases, particularly irregular, uncontrollable, incompressible traumatic bleeding. Therefore, it is important to develop hemostatic materials suitable for major bleeding.

Hemostatic materials are obtained by chemically modifying the active ingredient or processing it into a form. However, it remains a challenge to develop methods for providing active ingredients with more useful wound healing properties without affecting their hemostatic behavior. To date, many new hemostatic materials have been developed by chemically modifying or processing the active ingredient into a form, ranging from simple to complex, 2D to 3D, hydrophilic to hydrophobic, macroscopic to nanosize. The morphology of hemostatic materials affects their function and application, with the most widely used hemostatic material forms being sponges, hydrogels, nanofibers and granules or powders. In contrast, sponge materials are more attractive due to their shape recovery properties and liquid absorbing capacity, which may provide advantages for coping with some abnormal wounds. They can be injected in the form of a sponge in a compressed state, causing them to rapidly expand and fully contact the irregular bleeding wound. For example, zhang et al developed an injectable hemostatic sponge based on nanocellulose, which had the characteristics of high water absorption, fast shape recovery, strong antibacterial ability, and the like. Guo et al prepared a hybrid hemostatic sponge with high mechanical properties and excellent shape memory capabilities from polyvinyl alcohol, carboxymethyl chitosan and dopamine to address lethal incompressible bleeding. At present, the absorbent gelatin sponge is the most commonly used hemostatic material in clinic, belongs to non-toxic and non-antigenic protein gel substances, has a porous structure, and can absorb more than 30 times of the volume of the body of liquid and blood. Meanwhile, the gelatin sponge contains positron attachments, so that the gelatin sponge is favorable for adsorbing platelets to form small thrombus locally, and the hemostatic effect is achieved. However, the clinical use of the absorbent gelatin sponge also has the problems of low hemostatic efficiency, poor biocompatibility, weak mechanical properties and the like.

Shellac is a natural resin secreted by shellac, and has a molecular structure containing long-chain hydroxy fatty acid and sesquiterpene acid, so that the shellac has good film forming, tissue adhesion, biocompatibility and the like.

The invention takes bleached lac-lac and gelatin as raw materials to prepare the spongy multifunctional hemostatic material; and the material is compounded with polyphenol compound propyl gallate, so that the blood adsorption, hemostasis and antibacterial performance of the material can be further improved.

Disclosure of Invention

Aiming at the technical problems of low absorbability, low hemostatic efficiency, poor biocompatibility, weak mechanical property and the like of the existing clinical hemostatic sponge, the invention provides a hemostatic material and a preparation method thereof, wherein the hemostatic material has high fluid absorptivity, short hemostatic time and remarkably reduced blood loss; the water absorption rate and the water absorption rate are high, and the wound can be quickly plugged after water absorption; the liquid is absorbed quickly at the wound, the aggregation of platelets and red blood cells is accelerated, and the hemostasis is quick; the hemostatic material has good biocompatibility, antibacterial property and blood coagulation property, and has application prospects in biomedical applications such as wound blood coagulation hemostasis, tissue repair and the like.

To achieve the object of the present invention, the present invention provides a hemostatic material including gelatin and shellac.

Wherein, the weight ratio of the gelatin to the shellac is (0.05-1): 1) Preferably (0.2-0.3): 1, more preferably 0.2:1.

In particular, hemostatic enhancers are also included, which select gallic acid or gallic acid derivatives.

In particular, the gallic acid derivative is selected from propyl gallate, gallic acid, gallnut tannic acid, methyl gallate, lauryl gallate, cetyl gallate, stearyl gallate, and octacosanyl gallate, preferably propyl gallate and gallnut tannic acid.

In particular, the weight ratio of the hemostatic enhancer to the lac is (0.02-0.1): 1, preferably 0.04:1.

In particular, the weight ratio of propyl gallate and lac of the hemostatic enhancer is (0.02-0.1): 1, preferably 0.04:1.

In another aspect, the present invention provides a method for preparing a hemostatic material, comprising the steps of, in order:

1) Adding gelatin into lac solution, heating, stirring for dissolving, and preparing into lac-gelatin solution;

2) And (3) freeze-drying the lac-gelatin solution to obtain the hemostatic material.

In particular, the ratio of the volume of the shellac solution to the mass of gelatin in step 1) is 10: (0.05-1), preferably 10 (0.2-0.3), and more preferably 10:0.2.

In particular, the weight ratio of gelatin to lac in the lac-gelatin solution in the step 1) is (0.05-1): 1, preferably (0.2 to 0.3): 1, and more preferably 0.2:1.

In particular, the lac solution is a lac ammonia water solution and is prepared according to the following method: adding bleached lac into ammonia water solution, heating, stirring and dissolving.

In particular, the concentration of the aqueous ammonia solution is 0.1 to 0.5% by mass, preferably 0.2% by mass.

In particular, the mass volume concentration of the lac solution is 0.05-0.2g/mL, namely 0.05-0.2g, preferably 0.1g/mL of lac is dissolved in 1mL of ammonia water.

In particular, the heating temperature is 40-60℃and preferably 50 ℃.

Wherein, in the freeze-drying treatment process in the step 2), the temperature is controlled to be-50 to-10 ℃, preferably-40 ℃; the relative pressure is 20 to 70Pa, preferably 40Pa; the freeze-drying time is 60 to 80 hours, preferably 72 hours.

In another aspect, the present invention provides a method for preparing a hemostatic material, comprising the following steps in order:

1) Adding gelatin into lac solution, heating, stirring for dissolving, and preparing into lac-gelatin solution;

2) Adding a hemostatic enhancer into the lac-gelatin solution, stirring and uniformly mixing to prepare a lac-gelatin-enhancer mixed system, wherein the hemostatic enhancer is gallic acid or gallic acid derivatives;

3) And (3) performing freeze drying treatment on the lac-gelatin-enhancer mixed system to obtain the hemostatic material.

Wherein, the adding amount of the hemostatic enhancer in the step 2) is 20-100mg of hemostatic enhancer added into every 10mL of lac-gelatin solution, and preferably 40mg of hemostatic enhancer is added into every 10mL of lac-gelatin solution.

In particular, the hemostatic enhancer is selected from propyl gallate, gallic acid, gallnut tannic acid, methyl gallate, lauryl gallate, cetyl gallate, stearyl gallate, and octacosanyl gallate, preferably propyl gallate and gallnut tannic acid.

In particular, the hemostatic enhancer propyl gallate is added in an amount of 20-100mg of propyl gallate per 10mL of lac-gelatin solution, preferably 40mg of propyl gallate per 10mL of lac-gelatin solution.

In particular, the ratio of the hemostatic enhancer added in the step 2) to the shellac in parts by weight is (0.02-0.1): 1, preferably 0.04:1.

In particular, the weight ratio of propyl gallate and lac of the hemostatic enhancer is (0.02-0.1): 1, preferably 0.04:1.

In particular, in the freeze-drying treatment process in the step 3), the temperature is controlled to be between 50 ℃ below zero and 10 ℃ below zero, preferably 40 ℃ below zero; the relative pressure is 20 to 70Pa, preferably 40Pa; the freeze-drying time is 60 to 80 hours, preferably 72 hours.

The invention takes bleached lac-lac and gelatin as raw materials, prepares the spongy multifunctional hemostatic material through the crosslinking reaction of the bleached lac-gelatin and the gelatin, and is compounded with polyphenol compound propyl gallate to improve the blood adsorption, hemostasis and antibacterial performance of the material.

The hemostatic sponge has higher fluid absorptivity, excellent shape memory performance and stronger mechanical elasticity; in terms of biological performance, the whole blood coagulation index of the sponge is lower, and the aggregation capacity of blood cells and platelets is stronger; experiments of rat femoral artery injury models and liver capacity defect injury models show that the hemostatic time of the sponge is obviously less than that of the existing commercial gelatin sponge; in addition, the sponge has good antibacterial effect on staphylococcus aureus and escherichia coli, and has good biocompatibility and blood compatibility. The lac-gelatin composite spongy multifunctional hemostatic material has great application potential in cases of massive hemorrhage, and can meet the requirements of emergency treatment of hemorrhage in daily life and medical treatment.

Compared with the prior art, the invention has the following advantages:

1. the hemostatic sponge material disclosed by the invention has a rich hole structure, and is favorable for rapid entry and absorption of fluid, so that blood concentration and rapid thrombus formation are promoted.

2. The hemostatic sponge of the invention can be easily customized into various shapes to accommodate different wounds; repeated compression at greater pressures for 20 times still exhibits excellent shape memory function and mechanical properties, thus having great potential in narrow, irregular and incompressible wound applications.

3. The hemostatic sponge has excellent hydrophilic and hydrophobic synergistic effect, and can absorb about 10 times of the liquid in a very short time.

4. The hemostatic sponge has better blood coagulation capability and can well adhere erythrocytes and platelets; the hemolysis rate is lower than 3.5 percent, which accords with the application safety standard of the hemostatic sponge; in vivo and in vitro tests show that the sponge has good biocompatibility.

5. The hemostatic sponge has good hemostatic effect on femoral artery injury massive hemorrhage models, the hemostatic rate is better than that of the existing commercial gelatin sponge, and the sponge can also be used for hemostasis of internal organs such as liver and the like.

Drawings

FIG. 1 is an infrared spectrum of a hemostatic sponge;

FIG. 2 is an XRD pattern for a hemostatic sponge;

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

FIG. 4A is a graph of density test results of a sample of blood sponge;

FIG. 4B is a graph of the porosity statistics of the sponge sample;

FIG. 5 is a graph showing the results of compression set testing of SNPG sponge and gelatin;

FIG. 6A is a graph showing the volume expansion rate of the sponge after absorbing liquid;

fig. 6B is a photograph showing the different shapes of the SNPG0.2 sponge prepared in example 3;

FIG. 7A is a graph showing the volume recovery rate after the sponge absorbs the liquid and is squeezed;

FIG. 7B is a compression cycle chart of SNPG0.2 sponge prepared in example 3;

FIG. 7C is a compression cycle chart of SNPG0.6 sponge prepared in example 7;

FIG. 8A is a graph showing the results of the water absorption test of a sponge sample;

FIG. 8B is a graph showing the results of an absorption rate test of blood absorbed by a sponge sample;

FIG. 9 is a time measurement of the time required for complete absorption of a droplet on the surface of a blood sponge;

FIG. 10A is a BCI statistical plot of the hemostatic sponge;

fig. 10B is a graph of clotting time statistics for hemostatic sponges (where:, p <0.05;, p <0.01;, p < 0.001);

FIG. 11 is a statistical plot of hemostatic sponge versus erythrocyte adhesion;

FIG. 12 is an adhesion of red blood cells of a sponge sample, wherein A is a gauze sample; b is an SN sample; c is PG sample; b is SNPG0.2 sample; d is commercial gelatin sponge;

FIG. 13 is a statistical plot of platelet adhesion rate for a sponge sample of blood;

FIG. 14A is a graph showing the statistical result of the hemolysis rate of a sponge sample;

FIG. 14B is a photograph of hemolysis of a sponge sample; wherein a is positive control, b is SNG, c is SNPG0.1, d is SNPG0.2, e is SNPG0.3, f is SNPG0.4, g is SNPG0.5, h is PG, i is gauze, j is commercial gelatin sponge, and k is negative control;

FIG. 15 shows the viability of different sponge samples on NIH-3T3 cells;

FIG. 16 is a graph of stained sections of wound tissue 7 days after sponge sample implantation;

FIG. 16A is a photograph showing the inhibition of Staphylococcus aureus and Escherichia coli by sponge samples;

FIG. 16B is a statistical chart of the antibacterial rate of the sponge sample;

fig. 17A is a statistical graph (n.gtoreq.3) of bleeding amount during treatment of femoral puncture bleeding by hemosponge (wherein: p <0.05; p <0.01; p < 0.001);

fig. 17B is a statistical graph of bleeding stopping time (n.gtoreq.3) during treatment of femoral puncture bleeding by haemostatic sponge (where p <0.05; p <0.01; p < 0.001).

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Example 1

1. Preparing lac solution

Adding bleached lac into ammonia water solution, heating in water bath, stirring for dissolving, and preparing into ammonia water solution (SN solution for short), wherein the mass percentage concentration of the ammonia water solution is 0.2% (usually 0.1-0.5%); the mass volume concentration of the shellac ammonia water solution is 0.1g/mL (usually 0.05-0.2 g/mL), namely, 0.1g of shellac is bleached in each 1mL of ammonia water solution; the heating temperature in the water bath is 50 ℃ (usually 45-60 ℃);

adding 50.0g of bleached lac into 500mL of ammonia water solution with mass fraction of 0.2%, dissolving in water bath at 50deg.C for 1.5h to obtain 0.1g of lac ^-1 Shellac ammonia solution (named SN solution).

2. Preparing lac-gelatin mixed liquor

Adding gelatin into the prepared lac ammonia water solution (SN solution), heating, stirring, and dissolving to obtain lac-gelatin mixed solution; wherein the ratio of the mass of gelatin to the volume of the shellac ammonia water solution is 0.05:10 The ratio of the volume of the lac ammonia water solution to the mass of the gelatin is 10:0.05, namely 0.05g of gelatin is added to 10mL of the lac ammonia water solution, and the weight ratio of the gelatin to the lac in the lac-gelatin mixed solution is 0.05:1 (usually (0.05-1): 1), preferably (0.2-0.3): 1, and more preferably 0.2:1;

10mL of SN solution is measured respectively, gelatin with different quality is added into the SN solution, and the mixture is stirred and reacted for 2 hours at 50 ℃, and 0.05 g, 0.10 g, 0.20 g, 0.30 g, 0.40 g, 0.50 g and 0.60g of gelatin are added into each 10mL of SN solution respectively.

3. Preparing lac-gelatin-reinforcing agent mixed system

Adding propyl gallate (PG for short) serving as a hemostatic enhancer into the lac-gelatin mixed solution, stirring and uniformly mixing to prepare a lac-gelatin-enhancer mixed system (SNPG mixed system for short), wherein the adding amount of the propyl gallate serving as the hemostatic enhancer is 40mg propyl gallate (40-100 mg propyl gallate is usually added into 10mL of the lac-gelatin mixed solution, preferably 40 mg) per 10mL of the lac-gelatin mixed solution; the weight ratio of lac to propyl gallate in the lac-gelatin-gallic acid mixture is 1:0.04 (usually 1 (0.02-0.1), preferably 1:0.04).

The purpose of adding propyl gallate in the invention is to enhance the hemostatic and blood coagulation capabilities of the lac-gelatin sponge, and simultaneously enhance the antibacterial capabilities of the sponge and promote wound healing. After propyl gallate is added, the propyl gallate is connected in a lac-gelatin network through hydrogen bonding, so as to form a grid and hole structure.

The hemostatic enhancer is suitable for the present invention, except propyl gallate, other gallic acid, gallnut tannic acid, methyl gallate, lauryl gallate, cetyl gallate, stearyl gallate and octacosanyl gallate. The specific embodiment of the present invention will be described with reference to propyl gallate.

4. Freeze-drying treatment

And (3) performing freeze-drying treatment on the prepared lac-gelatin-gallic acid mixed system to obtain a hemostatic sponge (SNPG 0.05 sponge for short), wherein the temperature is controlled to be minus 40 ℃ and the pressure is controlled to be 40Pa for 72 hours in the freeze-drying process.

Respectively taking 3.0mL of a lac-gelatin-reinforcing agent mixed system, respectively placing the lac-gelatin-reinforcing agent mixed system in cylindrical grinding tools with the inner diameter of 20mm and the height of 20mm, placing a die in a freeze dryer, and performing freeze drying treatment, wherein the control process conditions of the freeze drying treatment are as follows: the freeze drying temperature is-40 ℃; the relative pressure is 40Pa; the freeze-drying time was 72h.

In the freeze-drying treatment process of preparing the hemostatic sponge, the temperature is controlled to be-50 to-10 ℃, and the preferable temperature is-40 ℃; the relative pressure is 20 to 70Pa, preferably 40Pa; the freeze-drying time is 60 to 80 hours, preferably 72 hours.

Examples 2 to 7

Except that the mass-to-volume ratio of the shellac-gelatin mixture in the step 2) was 0.10:10,0.20:10,0.30:10,0.40:10,0.50:10,0.60:10; the lac-gelatin-gallic acid mixed system prepared in the step 3) is respectively abbreviated as SNPG0.1, SNPG0.2, SNPG0.3, SNPG0.5 and SNPG0.6; the procedure of example 1 was repeated except that the hemostatic sponge obtained in step 4) was abbreviated as SNPG0.1, SNPG0.2, SNPG0.3, SNPG0.5, and SNPG0.6, respectively.

Comparative example 1

Except that the mass-to-volume ratio of the shellac-gelatin mixture in the step 2) is 0.2:10; the procedure of example 1 was repeated except that propyl gallate was not added to the lac-gelatin mixture, i.e., step 3) was not performed, and the sample group to which no PG was added was designated as SNG sponge.

Comparative examples 1A to 1E

Except that the mass-to-volume ratio of the shellac-gelatin mixture in the step 2) was 0.10:10,0.30:10,0.40:10,0.50:10,0.60:10; the procedure of example 1 was repeated except that propyl gallate was not added to the lac-gelatin mixture, i.e., step 3) was not performed, and the sample group to which no PG was added was designated as SNG sponge.

Comparative example 2

The ammonia water solution (SN solution) of shellac prepared in step 1) of example 1 was subjected to freeze-drying treatment by the same freeze-drying process as in step 4) of example 1 to prepare a sponge (SN sponge sample for short).

Test example 1 structural characterization of hemostatic sponge samples

1. Infrared spectrogram

The SNPG0.2 sponge prepared in example 3, the SNG sponge prepared in comparative example 1, the SN solid sample prepared in comparative example 2, gelatin, propyl gallate and lac were subjected to infrared spectrum detection, and the infrared spectrum is shown in FIG. 1.

Compared with lac, the SN solid sample is 1558cm ^-1 An expansion vibration absorption peak of C=N bond is added at the position, and the expansion vibration absorption peak is 3200cm ^-1 The absorption peak at the position becomes wide and strong, which indicates that the aldehyde group of the viologen is subjected to addition reaction with ammonia water when the viologen is dissolved in the ammonia water, and a product with an imine structure is generated.

GelatinThe molecule contains a large amount of carboxyl and amino, when the molecule and a small amount of propyl gallate are added into lac ammonia water solution for heating reaction for 2 hours, the produced SNPG sponge sample is 1654cm ^-1 And 1731cm ^-1 Two strong absorption peaks are arranged at the position, which indicates that chemical crosslinking reaction is generated between lac and gelatin, and a large number of amide bonds and ester bonds are generated; propyl gallate is added and then is connected into the lac-gelatin network through hydrogen bonding.

2. X-ray diffraction

The hemostatic sponges prepared in examples 2 to 4, the SN solid samples, and the gelatins used in examples 1 to 7 were subjected to X-ray diffraction using an X-ray diffractometer, and the crystal information of the samples was measured, and the measurement results are shown in fig. 2.

As shown in fig. 2, the diffraction peaks of the three sponge samples tested (i.e., the SNPG0.1, 0.2, 0.3 sponge samples) were substantially overlapping, with a broad amorphous peak at around 19.8 °. The narrowing of the peak width of SNPG compared to SN suggests that their aggregate morphology tends more toward a regular ordering structure.

3. Scanning by electron microscope

The morphology of the SN solid samples, the hemostatic sponge prepared in examples 2-7, and gelatin were observed with an electron microscope (SEM), and the observation results are shown in fig. 3. Apparent, the SN sample is loose, and the SN sample can scatter when lightly touched; the scan shows:

the SN sample is piled up into a continuous ditch ridge in the form of scales, and no obvious hole structure exists. When gelatin is added into an SN sample, all SNG sponges have hole structures, wherein the added amount of the gelatin in SNPG0.1 is small, and the hole structures are unevenly distributed and are easy to collapse and deform under the action of external force although the hole structures are formed; the SNPG0.2 and SNPG0.3 sponges exhibit interconnected macroporous structures with the pore size ranging from 212.5 to 425.3 μm. Subsequently, as the amount of gelatin added increases, the number of pores per unit volume of the SNPG0.4 and SNPG0.5 sponge of the SNPG sponge increases, but the average pore diameter gradually becomes smaller; the addition of gelatin is continuously increased, the sponge becomes compact, and only a few hole structures are seen. This is probably due to the increased amount of gelatin added, and the greater extent of crosslinking reaction of shellac with gelatin. As a control, commercial gelatin sponges showed a rich, inter-interlaced pore structure.

Test example 2 hemostatic sponge sample Density test

1.2 Density testing of sponge samples

3mL of SNPG samples prepared in the steps 1-7 and 3) of the example (namely a lac-gelatin-propyl gallate mixed system) are respectively injected into a cylindrical mold, and the SN solution prepared in the step 1) of the example 1 is injected into the cylindrical mold; and then freeze-drying to obtain a sponge test sample. The mass (m) of the sponge test sample is accurately weighed and measured respectively ₀ ) Diameter (R) and height (H), and the density of each test sample was calculated by formula (1):

the density test results are shown in fig. 4A, which shows that: as the amount of gelatin added in the SNPG increases, the density of the resulting sponge also gradually increases.

Test example 3 hemostatic sponge sample porosity test

The hemostatic sponges prepared in examples 1 to 7 were pre-weighed, and the weight (W ₀ G); then, each accurately weighed sponge is respectively soaked in deionized water for 30min, taken out, and the weight (W ₁ G); the volume (V, cm) of the hemostatic sponge after absorption was then determined ^-3 ) The method comprises the steps of carrying out a first treatment on the surface of the The porosity (%) of the hemostatic sponge is then calculated according to formula (2).

In formula (2): w (W) ₀ And W is ₁ The weight (g) of the sponge before and after soaking is respectively reached; ρ is the density of water (1.0 g.cm) ^-3 ) The method comprises the steps of carrying out a first treatment on the surface of the V is the volume of the blood sponge after water absorption. All hemostatic sponge sample measurements were repeated five times.

In the sample preparation process, a circular grinding tool with the inner diameter of 20mm and the height of 20mm is adopted, so that the prepared sample is in a cylindrical state, and the diameter R and the height H of the water absorbed sample are measured by a vernier caliper to obtain the volume V of the sponge sample after water absorption.

The results of the hemostatic sponge porosity measurement are shown in fig. 4B, and fig. 4B shows that the change trend of the porosity of the SNPG sponge is opposite to the density, and the porosity gradually decreases with the increase of the gelatin addition amount. The porous structure of the sponge facilitates rapid entry and absorption of fluid, while the appropriate pore structure size facilitates accumulation of components in the blood in the pore structure to create a thrombotic structure, thereby promoting clotting of the blood. On the one hand, when the surface hole structures of the hemostatic sponge are all closed, blood cells in blood are not easy to enter. On the other hand, when the diameter of the hole structure of the hemostatic sponge is too large, more blood cells are needed to form the micro-thrombus structure, which is not beneficial to the efficiency of the whole thrombus formation. Finally, the hemostatic sponge can reach the optimal state only when the composite sponge simultaneously has the surface hole structures which are mutually communicated and holes with proper sizes, and the hemostatic sponge can achieve the purposes of rapid hemostasis by concentrating blood and forming thrombus structures. Thus, in combination with SEM results and porosity analysis, the SNPG0.2 and SNPG0.3 sponges possess optimal pore structures.

Test example 3A mechanical Property (compression set) test of hemostatic sponge

Compression testing was performed on hemostatic sponges prepared in examples 1-6 and on purchased sponges (spirot absorbable gelatin sponge wound care sponges medical dental oral material surgical emergency wound bleeding gel swelling sponge patches, type B: 20mm x 20 mm) using a TMS-Pro physical analyzer (FTC limited in the united states).

Before testing, the diameter and height of each sponge sample were accurately recorded, and then the sponge samples were soaked in deionized water for 30min to allow adequate water absorption. In the test, the sponge was placed horizontally on a test bench, the compression rate was set at 10.0mm/min, and the pressure, strength and sample morphology required for the samples at deformation rates of 40%, 50% and 70%, respectively, were recorded. Each set of samples was tested 5 times. The measurement results are shown in FIG. 5.

As shown in fig. 5, under three different deformation conditions, the compressive stress of the SNPG sponge gradually increases with increasing gelatin content, because the more gelatin, the greater the degree of crosslinking of shellac and gelatin, the greater the amount of polymer formed and the greater the density, and therefore the greater the stress required. Meanwhile, the test result shows that under the condition that the deformation rate is 40% and 50%, all SNPG sponges tested can keep good form at the end of the test; however, at a deformation rate of 70%, the morphology of the other SNPG sponge was changed greatly except for the SNPG0.2 and SNPG0.3 samples, wherein the SNPG0.05 sample with a small gelatin addition amount was partially dissolved, the sponge of SNPG0.1 collapsed into an amorphous state, and the SNPG0.4, SNPG0.5 and SNPG0.6 with a large gelatin addition amount had cracking phenomenon under pressure, and the more gelatin content was, the more cracks were formed.

Test example 3B Water-triggered shape memory Capacity test of hemostatic sponge

Sponges with shape memory properties have particular advantages in hemostasis, such as for stenotic, irregular and incompressible wounds. To evaluate the water-triggered shape memory ability of the SNPG sponge, the volume expansion rate of the sponge was measured as follows:

the volume expansion rate testing method comprises the following steps: original diameter R and original height h of the sponge sample are measured by a vernier caliper, and original volume of the sponge is calculated to be V according to a cylinder volume formula ₀ . Soaking the sponge sample in deionized water at 25deg.C for 30min, and measuring diameter R of the sponge sample after water absorption with vernier caliper ₁ And a post-water absorption height h ₁ Then calculate the volume V after water absorption ₁ Then V ₁ And V is equal to ₀ The ratio of (2) is the volume expansion rate of the sponge, and the test result is shown in FIG. 6A.

To illustrate that the sponge sample can be prepared in various forms, irregular wounds of the body can be dealt with. The sponge sample prepared in example 3 of the present invention was cut into various forms and absorbed with water, and the shape of the absorbed sponge sample was as shown in fig. 6B.

As can be seen from fig. 6A: the SNPG sponge shows a certain volume expansion after absorbing the liquid, the volume expansion rate of which decreases with increasing gelatin addition, wherein there is no significant difference in the volume expansion rate of the SNPG0.05 to SNPG0.2 sponge, whereas the volume expansion rate of 26.1% of the SNPG0.3 sponge is significantly less than 44.4% of the SNPG0.2 sponge, probably because the pore structure of the sponge decreases with increasing gelatin amount. The good volume expansion can enable the hemostatic sponge to fully absorb liquid components in blood, so that the concentration of local blood cells and platelets is increased, and the hemostatic sponge is beneficial to the formation of a thrombus structure. On the other hand, the volume expansion of the hemostatic sponge after absorbing the liquid is beneficial to the local compression effect, so that the aim of further hemostasis is achieved. In addition, SNPG0.2 can be easily customized to accommodate different wounds (FIG. 6B), and the volume expansion rate of the sponge after absorbing liquid is maintained between 40.2 and 56.3 percent.

Test example 3C test of the volumetric recovery Performance of hemostatic sponge after liquid suction extrusion

Volume recovery rate: the sponge samples prepared in examples 1-7 were respectively soaked in deionized water for 30min, and the diameter R of the sponge sample after water absorption was measured by using a vernier caliper ₁ And a post-water absorption height h ₁ Calculating the initial volume V of the immersed water according to a cylinder volume formula ₁ . After the measurement, the sponge is placed on a 100g weight and pressed for 1h, the sponge is soaked in deionized water again for 30min, and then the diameter R of the sponge after water absorption is measured again ₂ And height h ₂ Calculating the volume V of the sponge re-immersed water according to the volume formula of the cylinder ₂ Then V ₂ And V is equal to ₁ The ratio of the two is the recovery rate of the sponge. The test results are shown in FIG. 7A.

As shown in fig. 7A, the volume recovery rate of the other sponges was 92% or more, except that SNPG0.5 was not recovered to the original state due to partial dissolution.

Test example 3D test of the cyclic compression Performance of hemostatic sponge

The compression set-recovery ability, i.e., the cyclic compression performance, of the sponge was examined using a TMS-Pro physical property analyzer (FTC Co., ltd.).

The SNPG0.2 sponge prepared in example 3 and stored for 6 months, and the SNPG0.6 sponge prepared in example 7 were immersed in deionized water for 30 minutes to fully absorb water, and then subjected to a cyclic compression test using a physical property analyzer. In the experimental process, the sponge sample was allowed to fully absorb water before the next compression was started after each compression was completed. The diameter and height of the sample were accurately recorded each time for calculation. During testing, the sample is horizontally placed on a test bench, the compression rate is set to be 10.0mm/min, the deformation rate is set to be 50%, and the cycle number is 20. The test results are shown in FIGS. 7B and 7C.

As can be seen from fig. 7B and 7C, the SNPG0.2 sponge shows excellent shape memory function even after being repeatedly compressed for 20 times after being stored for 6 months, and the strain of the SNPG0.6 sponge with a large gelatin addition amount is continuously reduced with the increase of the compression times (as shown in fig. 7C). Thus, by comprehensive analysis, the mechanical properties of SNPG0.2 were optimal in all sponges.

Test example 4 liquid absorption Property test of hemostatic sponge sample

The liquid absorption performance test of the sponge sample is carried out by referring to the medical industry standard YY/T0471.1-2004 part 1 liquid absorption of the contact wound dressing test method.

The hemostatic sponges prepared in examples 1 to 6 and the samples prepared in comparative examples 1 and 2 were each previously cut into a certain volume of small sponge samples, and their weights (denoted as W were measured respectively ₀ ) Then immersing the small sample of the cut sponge sample into 15mL deionized water and 15mL rat blood (diluted 10 times with physiological saline) containing sodium citrate anticoagulant respectively at 25deg.C and 37deg.C for 30min, holding one corner of the sponge sample after sufficient solution absorption with forceps, suspending for 30s, and weighing the absorbed sponge sample (denoted as W ₁ ). The liquid absorption rate of the sponge sample was calculated according to formula (3).

The test results are shown in FIGS. 8A and 8B. SN without gelatin is almost completely dissolved in water and blood, and has no ability to absorb liquid. As shown in fig. 8A, the SNPG sponge prepared by adding gelatin to SN has water absorption, wherein 0.05 of SNPG is dissolved by a little solid, and the water absorption at room temperature is 961.9%; the water absorption rate of SNPG0.1 is highest in the prepared sponge and is 1247.9%; thereafter, as the amount of gelatin added in the SNPG increases, the water absorption of the sponge gradually decreases. In accordance with the law of change in water absorption, as shown in fig. 8b, the blood absorption capacity of the SNPG sponge also showed that the blood absorption rate increased and then decreased with the increase of the gelatin addition, wherein the blood absorption rate of SNPG0.1 was 979.4%. Meanwhile, the test result of temperature versus absorption performance shows that the water absorption rate and the salt solution absorption rate of the SNPG sponge sample are higher than those of the room temperature in the normal body temperature of 37 ℃, probably because the pores of the sponge become larger due to the increase of the temperature, so that the liquid absorption capacity is enhanced.

Test example 4A test of the liquid absorption rate performance of hemostatic sponge sample

The time required for the deionized water droplets to be absorbed by the sponge materials prepared in examples 1-6 was recorded using the sessile drop function test of the contact angle meter. The test results are shown in FIG. 9.

As shown in fig. 9, the diffusion absorption time of the liquid drops on the surfaces of the SNPG0.05, SNPG0.1 and SNPG0.2 sponges is within 1 second, and the hemostatic sponge of the invention can absorb liquid blood rapidly and can absorb liquid blood rapidly; with increasing gelatin addition in the sponge, the time required for the liquid drop to completely diffuse and be absorbed gradually prolonged, and the time required for the liquid drop on the surface of the SNPG0.5 sponge to completely diffuse and be completely absorbed is 27s.

As a control, the commercial gelatin sponge had a water contact angle of 141.05 °, exhibited significant hydrophobicity, and the time required for the droplet to completely diffuse and absorb on its surface was greater than 5min without pressing the commercial gelatin sponge. The experimental result shows that the SNPG sponge prepared by the invention has proper hydrophilic and hydrophobic properties, can absorb a large amount of blood in a very short time, has excellent liquid-triggered shape memory capacity, and has great application potential in the aspect of massive hemorrhage caused by trauma or operation. Test example 5 in vitro coagulation test of hemostatic sponge sample

The in vitro clotting properties of the SNPG sponge were evaluated using the clotting index (BCI).

Firstly, 50.0mg of each hemostatic sponge sample prepared in examples 2-6 is weighed and placed in each caseThe glass culture dish; meanwhile, medical gauze, commercial gelatin sponge, and SNG sponge prepared in comparative example 1 of the same weight were set as controls. Accurately weighing 9.0mL of citric acid rat whole blood, adding 1.0mL of CaCl with concentration of 0.2mol/L ₂ The solution activated rat blood, and 100uL of activated rat blood was dropped onto the sample surface, respectively. 100uL of rat blood was dissolved in 10mL deionized water as a blank sample group. After incubating the above samples at 37℃for 3min, 10mL of deionized water was added, respectively, and incubated in an oven at 37℃for another 10min, and then the samples were taken out and the non-coagulated blood was rinsed with deionized water. The rinse was collected and the absorbance of the supernatant measured at 540 nm. All experiments were repeated 3 times. The coagulation index (BCI) was calculated according to formula (4), and the result is shown in fig. 10A:

900uL of citric acid rat blood was added with 100uL of 0.2M CaCl ₂ Solution, activated rat whole blood. Sponge (30 mg) was accurately weighed into a tube, 200. Mu.L of citric acid rat whole blood was added, the tube was tilted every 10s, the fluidity of the blood was observed, and the blood clotting time was recorded. Each sample was repeated three times and the results recorded as in fig. 10B.

As shown in fig. 10A, all the SNPG sponges had a clotting effect, with the lowest coagulation index value of SNPG0.2 being 10.5%, which is only half that of the SNG sponges; as shown in FIG. 10B, SNPG0.2 sponge was also the fastest in all samples tested, with a clotting time of only about 45s, whereas SNG sponge had a clotting time of 70s. As a control, the BCI values of gauze and commercial gelatin sponge groups were greater than 85% and the clotting times were 324s and 270s, respectively, indicating that they were less effective. The results show that the addition of PG in proper amount helps to improve the coagulation effect of SNPG sponge

Test example 6 in vitro adhesion test of erythrocytes of hemostatic sponge

Fresh rat blood was centrifuged at 1000rpm for 10min and plasma was removed. The hemostatic sponge (SNPG 0.2) prepared in example 3 was cut into 0.5 cm. Times.0.5 cm, 50. Mu.L of the erythrocyte suspension was dropped on the surface of the sponge, and after culturing at 37℃for 10 minutes, non-adherent erythrocytes were removed by washing with PBS. Then, all samples of adhered erythrocytes were transferred to 1.0mL of deionized water, and adhered erythrocytes were lysed at 37℃for 1h, and the absorbance of the resulting solution was measured at 540 nm.

In addition to using hemostatic sponge (SNPG 0.2), erythrocyte adhesion test was performed using gauze, commercial gelatin sponge, SNG sample prepared in comparative example 1, SN sample prepared in comparative example 2, propyl Gallate (PG).

The blank control group (also called reference group) was a suspension of cells of fuchsin without sponge, absorbance was measured, and each group of samples was measured three times. The adhesion rate (%) of erythrocytes was calculated according to formula (5): the results of the erythrocyte adhesion rate test are shown in FIG. 11.

Meanwhile, the sponge to which the red blood cells were adhered was fixed with 2.5% glutaraldehyde for 24 hours, and then dehydrated with a series of graded ethanol solutions of 50%,70%,80%,90%,100% for 20 minutes, and the sponge was observed with a scanning electron microscope, and the observation results are shown in fig. 12.

The results of the erythrocyte adhesion test are shown in fig. 11. The red blood cell adhesion rates of gauze and commercial gelatin sponge are 19.1% and 7.5%, respectively, which are significantly lower than that of SNPG sponge. SN samples without added gelatin and propyl gallate also showed 54.9% erythrocyte adhesion, which may benefit from SN carrying a positive charge in the form of an ammonium salt with electrostatic interactions between erythrocytes. The adhesion rate of the propyl gallate PG to erythrocytes was 55.2%, which is equivalent to that of SN of comparative example 2, probably because the phenolic hydroxyl groups in the molecular structure thereof were chemically reacted with proteins. However, the red blood cell adhesion rates of SNG and SNPG0.2 were 86.8% and 90.9%, respectively, significantly higher than the other groups, but there was no significant difference between the two, indicating that the crosslinking reaction of shellac and gelatin and the network structure formed greatly promoted the rapid adhesion of red blood cells and concentration of blood cells.

The condition of the hemostatic material adsorbing red blood cells was further observed by scanning electron microscopy, and the result is shown in fig. 12. Only a few red blood cells on the gauze are distributed on the long fiber (graph a); the SNPG0.2 still remained the original continuous pore structure after absorbing blood, and many red blood cells adhered and accumulated in the pores irregularly, and most cells were observed to maintain the sphere morphology (panel B); as a control, propyl gallate powder also had better adhesion of erythrocytes, which accumulated on the surface and interstices of its powder (panel C), but few erythrocytes adsorbed on commercial gelatin sponge (panel D).

Test example 7 platelet adhesion test of hemostatic sponge

Freshly obtained rat blood was centrifuged at 1000rpm for 10min, platelet Rich Plasma (PRP) was collected and subjected to a platelet adhesion test.

The hemostatic sponge (SNPG 0.2) prepared in example 3 was cut to a size of 0.5 cm. Times.0.5 cm; 50. Mu.L of PRP plasma was then added dropwise to each sponge sample, incubated at 37℃for 30min, and the samples were rinsed with PBS to remove non-adherent platelets. The attached platelets were then lysed with 1.0mL of a 1.0% Triton X-100 solution at 37℃for 1h, followed by detection of platelet lactate dehydrogenase activity using the LDH Kit (Kit) as determined according to the Kit instructions.

Platelet adhesion tests were performed using gauze, commercial gelatin sponge, SNG sample prepared in comparative example 1, SN sample prepared in comparative example 2, propyl Gallate (PG), in addition to hemostatic sponge (SNPG 0.2).

The absorbance was read with a microplate reader at 450nm using 50 μl of plasma without sample contact as a reference group (also called a blank group). Platelet adhesion rate was calculated according to equation (6) and the test results are shown in fig. 13.

Platelet adhesion test results are shown in fig. 13: overall, the tested samples showed substantially consistent platelet adhesion to red blood cells, with platelet adhesion rates of SNG and SNPG0.2 as high as 87.6% and 90.8%, significantly higher than other groups, with only 18.3% and 20.2% for the control gauze and commercial gelatin sponge.

Test example 8 in vitro haemostatic sponge sample haemolysis test

Rat blood is collected in a test tube containing sodium citrate anticoagulant, 1.0mL of primary blood is taken out after being sufficiently and evenly shaken, and is diluted with 5.0mL of physiological saline, so as to obtain a blood sample for standby. The hemostatic sponges prepared in examples 2-6 were weighed 1.0mg each, incubated with 1.0mL of physiological saline at 37℃for 12h, respectively, to prepare a sponge sample suspension, and filtered, leaving a filtrate. 100. Mu.L of diluted blood was added to the filtrate and incubated at 37℃for 1 hour, followed by centrifugation at 3000rpm for 10 minutes, and photographs were taken. The supernatant was collected, and the absorbance of each liquid at 540nm was measured.

A hemolysis test was performed using SNG samples, propyl gallate powder, gauze and commercial gelatin sponge samples prepared in comparative example 1 as controls.

The pure water and physiological saline without sponge sample were used as positive and negative controls, respectively, and the hemolysis ratio (%) was calculated according to formula (7):

the test results are shown in fig. 14A and 14B, and the safety standard of the haemostatic material with the haemolysis rate is less than 5% according to national standard requirements. The haemostatic sponge was investigated for haemocompatibility by haemolysis experiments. As shown in fig. 14B, almost all red blood cells of the blood in pure water (positive control group) ruptured to release hemoglobin, and the solution was bright red; while SNPG sponges and other controls showed blood clotting, with the supernatant being lighter in color or even colorless. By measuring the hemolysis rate (fig. 14A), the sample hemolysis rate was lower than 3.5%, which meets the application safety standard of hemostatic sponge. As can be seen from fig. 14A, the hemolysis rate of the SNPG0.1 to 0.5 sponge to which propyl gallate was added was lower than that of the SNG to which propyl gallate was not added, indicating that the addition of propyl gallate to the sponge could reduce the hemolysis rate.

Test example 9 in vitro cell compatibility test of hemostatic sponge

Cell expansion culture: NIH-3T3 cells (purchased to Guangzhou Hua Tuo organism) stored in a frozen liquid nitrogen tank are quickly thawed in a water bath at 37 ℃, and after complete thawing, DMEM culture solution containing 10% FBS+1% Ps is added into the cells in time to resuspend the cells. Transferring melted cell liquid into 15mL centrifuge tube in sterilized super clean bench, centrifuging at 1000rpm for 5min on centrifuge, discarding supernatant, adding 1.0mL culture medium to resuspend cell, transferring into cell culture bottle, supplementing culture liquid to 6.0mL, adding 5% CO ₂ Culturing at 37deg.C, and changing liquid once every other day. When the cells grow to a certain density, the cells are passaged at 1:2 or 1:3 at 5% CO ₂ Culturing at 37deg.C.

CCK8 method for detecting influence of material on proliferation activity of NIH-3T3 cells

The cells in the logarithmic phase were grown in 1X 10 ⁶ Each group was inoculated into 96-well plates, 100uL of each well was inoculated, after cell adhesion, materials (sponge prepared in example 3, sponge prepared in comparative example 1) of different concentrations (0, 10,20,30,40,50,60 ug/mL) were added to the cell culture system, 5 complex wells were set up for each group, 10uL of CCK8 reagent was added to each well 24h after treatment, incubation was performed for 3h at 37 ℃, absorbance values at a measurement wavelength of 450nm were taken out, cell viability was calculated according to formula (8), and statistical analysis results were shown in fig. 15.

Wherein: as, absorbance of experimental group (containing cells, medium, CCK-8 solution and drug solution); ac, absorbance of control (cell, medium, CCK-8 solution, drug-free); ab, absorbance of blank (medium, CCK-8 solution, cell, drug free).

To examine the biocompatibility of the sponge, the toxicity of the sponge extract on mouse embryonic fibroblasts (NIH-3T 3) was tested. The CCK-8 assay showed that the viability of SNG and SNPG0.2 cells at different concentrations exceeded 80% after 24h incubation at different sample concentrations, indicating that the sponge samples were non-cytotoxic, as shown in FIG. 15.

Test example 10 in vivo biocompatibility test of hemostatic sponge

Before the experiment, the sponge sample prepared in example 3, the sponge sample prepared in comparative example 1, commercial gelatin sponge, and Propyl Gallate (PG) powder were prepared into test pieces of the same shape and size (diameter 10mm, height 5 mm), and were sterilized under ultraviolet irradiation for 3 hours, respectively.

The 15 male SD rats were randomly divided into 5 groups, a blank group, a PG sample group, a commercial gelatin sponge group, a sponge SNG group, and a sponge SNPG group, each of which was 3. All rats were anesthetized with 10% chloral hydrate solution (1.2 mL/250 g) intraperitoneal injection. Subsequently, a small incision was created in the back area of each rat. Each prepared sample was implanted into the incision and the skin was then closed. No sample cuts were taken as blank. After 7d the rats were sacrificed and test samples and adjacent tissues were excised. The obtained material, tissue samples, were paraffin embedded, sectioned and attached to glass slides. Acute and chronic inflammatory responses were detected using hematoxylin-eosin staining. The stained sections were observed and analyzed under a microscope. Staining section of wound tissue 7 days after sponge sample implantation, as shown in fig. 16.

As can be seen from fig. 16: the prepared sponge sample was implanted into a rat back incision, and then the skin was closed. After 7 days of sample implantation, the wound tissues of rats in blank groups, SNG, SNPG0.2 and PG groups have almost no inflammatory reaction, and a large number of new blood vessels exist in the tissues, which indicates that the SNG and SNPG sponges have good in-vivo compatibility and have potential in-vivo application as biocompatible hemostatic agents. As a control, commercial gelatin sponge was implanted with more inflammatory cells in rat tissue.

Test example 11 antibacterial property test of hemostatic sponge

The good antibacterial property can prevent wound infection, and the inhibition effect of the sponge on staphylococcus aureus and escherichia coli is examined.

Separating staphylococcus aureus (ATCC 25923) and escherichia coli (ATCC-25922) and preparing 10 respectively ⁸ Cfu suspensions were used to determine the in vitro bacteriostatic activity of the SN, SNG, SNPG 0.2.2 and PG four groups of samples. Respectively accurate5.0mg of the sponge samples prepared in comparative examples 1 and 2 and example 3 and propyl gallate PG were weighed into 20mL centrifuge tubes, and 1mL of Luria-Bertani liquid medium and 10 mL of the mixture were added, respectively ⁸ Cfu suspensions were used as a blank with Luria-Bertani broth without added samples. Centrifuge tubes were incubated at 37℃for 4 hours under aerobic conditions, and a shaking table was used to ensure uniform distribution at 160 rpm. After incubation, the suspension was diluted 10 in PBS buffer ⁵ After incubation of 30uL of the suspension Luria-Bertani solid medium for 24h at 37 ℃ under aerobic conditions, single colonies were counted using the spread plate method, as shown in fig. 16A, 16B. The SNG and the SNPG0.2 have certain inhibition effects on two bacteria, wherein the inhibition rates of the SNPG0.2 sponge on staphylococcus aureus and escherichia coli are respectively 72.9% and 80.4%, and the inhibition effects are better than those of the SNG. This is because the PG component added in SNPG0.2 has a remarkable antibacterial effect, and the antibacterial rate against escherichia coli exceeds 99.9%. The results show that the addition of PG in the sponge sample can not only accelerate the coagulation efficiency, but also improve the antibacterial performance.

Test example 12 in vivo hemostatic Performance test of hemostatic sponge

Rat femoral artery lesions were used as a major bleeding model to evaluate hemostatic performance of gauze, SNG, SNPG0.2 and commercial gelatin sponge in vivo.

The hemostatic performance of the sponges was evaluated by establishing a femoral artery injury model using SD male rats. Prior to the experiment, all male rat abdominal cavities were anesthetized with 10% chloral hydrate solution (1.2 mL/250 g) and fixed on surgical cork board. When the femoral artery is damaged, the thigh hair of the rat is scraped off, and the inguinal muscle is cut off to expose the femoral artery. The wound was exposed for 5s with a 20G needle puncture on the femoral artery to ensure normal bleeding. Immediately thereafter, a hemostatic sample of size 10mm in diameter and 5mm in height was applied to the bleeding site, and the wound was manually pressed with a medical gauze to stop bleeding, wherein the hemostatic sample was (gauze, sponge sample prepared in comparative example 1, example 3, commercial gelatin sponge; hemostatic time was recorded until the wound was no longer bleeding after the sponge was removed, and the test results were shown in fig. 17A, 17B.

As shown in fig. 17A, SNG and SNPG0.2 immediately controlled strong bleeding compared to gauze and commercial gelatin sponge, and the average amount of bleeding in rats they treated was 345.7mg and 251.6mg, respectively; the bleeding amount of NPG0.2 treatment group was significantly less than that of SNG group (p < 0.05); and both SNG and SNPG0.2 were significantly lower than the gauze and commercial gelatin sponge sets 801.0mg and 652.7mg (p < 0.001).

As shown in fig. 17B, SNG and SNPG0.2 also required significantly less hemostasis time than the other two groups from the hemostasis time point of view. Notably, the hemostasis time of SNPG0.2 added with propyl gallate is 107s, the hemostasis time of SNG is 183s, and certain difference (p < 0.01) exists between the two; the amount of bleeding in the SNPG0.2 treated group was also significantly less than in the SNG group.

The experimental result shows that the SNPG0.2 sponge has good hemostatic effect on femoral artery injury massive hemorrhage model and even has obvious advantages compared with the gelatin sponge on the market, so the SNPG0.2 sponge has great application potential in the fields of daily injury, surgical first aid and the like.

The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Claims (10)

1. A hemostatic material is characterized by comprising gelatin and shellac.

2. The hemostatic material of claim 1, wherein the weight ratio of gelatin to shellac is (0.05-1): 1) Preferably (0.2-0.3) -1, more preferably 0.2:1.

3. The hemostatic material of claim 1 or 2, further comprising a hemostatic enhancer selected from gallic acid or a gallic acid derivative.

4. A hemostatic material according to claim 3, wherein the gallic acid derivative is selected from propyl gallate, gallic acid, gallnut tannic acid, methyl gallate, lauryl gallate, cetyl gallate, stearyl gallate and octacosanyl gallate.

5. A haemostatic material according to claim 3, wherein the ratio of the haemostatic enhancer to the shellac is (0.02-0.1) to 1, preferably 0.04:1.

6. The preparation method of the hemostatic material is characterized by comprising the following steps in sequence:

1) Adding gelatin into lac solution, heating, stirring for dissolving, and preparing into lac-gelatin solution;

2) And (3) freeze-drying the lac-gelatin solution to obtain the hemostatic material.

7. The method of claim 6, wherein the ratio of the volume of the shellac solution to the mass of the gelatin in step 1) is 10: (0.05-1), preferably 10 (0.2-0.3), and more preferably 10:0.2.

8. The preparation method of claim 6 or 7, wherein the shellac solution is shellac ammonia water solution, and is prepared by the following steps: adding bleached lac into ammonia water solution, heating, stirring and dissolving.

9. The preparation method of the hemostatic material is characterized by comprising the following steps in sequence:

1) Adding gelatin into lac solution, heating, stirring for dissolving, and preparing into lac-gelatin solution;

2) Adding a hemostatic enhancer into the lac-gelatin solution, stirring and uniformly mixing to prepare a lac-gelatin-enhancer mixed system, wherein the hemostatic enhancer is gallic acid or gallic acid derivatives;

3) And (3) performing freeze drying treatment on the lac-gelatin-enhancer mixed system to obtain the hemostatic material.

10. The method of claim 9, wherein the hemostatic enhancer in step 2) is added in an amount of 20-100mg of hemostatic enhancer per 10mL of shellac-gelatin solution, preferably 40mg of hemostatic enhancer per 10mL of shellac-gelatin solution.