Multifunctional hemostatic sponge as well as preparation method and application thereof

Abstract

The invention relates to a multifunctional hemostatic sponge as well as a preparation method and application thereof. The hemostatic sponge comprises a matrix formed by cross-linking methylacryloylated collagen and nano-hydroxyapatite modified by epigallocatechin gallate, the mass ratio of the methacrylated collagen to the nano-hydroxyapatite modified by the epigallocatechin gallate is (2 to 30): 1. By combining ColMA and HAP coated EGCG and optimizing the proportion, the obtained hemostatic sponge has a good collagenase degradation resisting effect and hemostatic, anti-inflammatory and antibacterial effects, tissue regeneration and repair can be promoted, the use comfort of a patient is higher, and the treatment effect is better.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a multifunctional hemostatic sponge and a preparation method and application thereof. Background Art

[0002] Local bleeding and wound healing are common clinical challenges during dental procedures such as oral surgery, tooth extraction, and dental implants. Because the oral environment is moist, contains many bacteria, and is rich in enzymes, traditional hemostatic materials have obvious deficiencies in hemostatic effects, antibacterial and anti-inflammatory properties, and healing functions. In addition, the oral structure is complex, and it is difficult to reach hidden areas of the wound when injecting or applying materials. Therefore, it is necessary to develop a material with rapid hemostasis, antibacterial and anti-inflammatory properties, and good biocompatibility to improve the quality of oral postoperative care.

[0003] Currently, the hemostatic materials used in the oral cavity mainly include medical gauze, gelatin sponge, chitosan-based materials, etc. Although these materials have certain effects in hemostasis, they still have some limitations:

[0004] (1) Short-term hemostatic effect: Existing materials usually only have a short-term hemostatic effect and are difficult to meet the needs of long-term hemostasis.

[0005] (2) Limited antibacterial ability: The oral environment is complex and contains a large number of pathogenic bacteria, such as Porphyromonas gingivalis and Streptococcus. Most traditional hemostatic materials lack effective antibacterial function and are prone to cause wound infection.

[0006] (3) Insufficient anti-inflammatory and healing effects: Inflammatory reactions after surgery are common, but existing materials mostly focus on hemostatic function and lack the multifunctionality of anti-inflammatory and promoting tissue healing.

[0007] (4) Poor biocompatibility: Some synthetic materials are prone to cause local inflammation or allergic reactions, reducing patient comfort and treatment effects.

[0008] Therefore, there is an urgent need to develop a hemostatic material that combines rapid hemostasis, antibacterial and anti-inflammatory effects, and promotion of tissue regeneration, which can meet the clinical needs in the complex oral environment and have good operational convenience and biocompatibility. Summary of the invention

[0009] In view of the deficiencies in the above-mentioned prior art, the present invention proposes a novel multifunctional hemostatic sponge and a preparation method thereof, which can be used for oral hemostasis and has the functions of rapid hemostasis and antibacterial and anti-inflammatory functions.

[0010] The specific technical scheme of the present invention is as follows: a multifunctional hemostatic sponge ColMA / HAP@EGCG, comprising a matrix cross-linked by methacryloylated collagen (ColMA) and nanohydroxyapatite (HAP@EGCG) modified with epigallocatechin gallate, wherein the mass ratio of ColMA to HAP@EGCG is 2 to 30:1.

[0011] Hydroxyapatite (HAP) has a hemostatic effect, and EGCG (epigallocate) has good antibacterial properties, which helps to control inflammatory reactions. The present invention cross-links and compounds methacrylylated collagen (ColMA) with EGCG-modified HAP, and HAP provides a sustained-release platform for EGCG. The three components cooperate and support each other, so that the hemostatic material has good biocompatibility, stable structure, higher strength, better hemostatic performance, and also has good anti-inflammatory and antibacterial effects, can promote tissue regeneration and repair, and promote healing. In particular, it can maintain an effective physical form for a long time in a high-humidity and high-enzyme environment in the oral cavity, has a long action time, and is more comfortable for patients to use and has a better therapeutic effect.

[0012] Preferably, the epigallocatechin gallate modified nanohydroxyapatite is obtained by adding nanohydroxyapatite to the epigallocatechin gallate solution, dispersing, stirring, mixing, reacting, washing, and drying at 40-50° C. The epigallocatechin gallate solution can be prepared by dissolving epigallocatechin gallate in deionized water or a suitable solvent (such as aqueous ethanol). The drying method can be heating drying or freeze drying, preferably freeze drying, which has better dispersibility.

[0013] Preferably, the concentration of the epigallocatechin gallate solution is 1-10 w / v%, and the mass ratio of epigallocatechin gallate: nano-hydroxyapatite is 0.2-0.5:1.

[0014] Preferably, the methacryloyl collagen is methacryloyl type I collagen.

[0015] Further preferably, the methacryloyl collagen is prepared by adding methacrylic anhydride dropwise to a type I collagen aqueous solution at pH 7, and reacting at room temperature, wherein the collagen: water: methacrylic anhydride = 5 g: 500 mL: 10 mL.

[0016] Preferably, after the reaction, dialysis is performed using a 3500Da dialysis bag, and the centrifugal speed is 8000-15000 rpm.

[0017] Another aspect of the present invention provides a method for preparing the hemostatic sponge, comprising the steps of: adding nano-hydroxyapatite modified with epigallocatechin gallate to a methacrylylated collagen solution containing a photoinitiator, mixing evenly to obtain a mixed solution, then subjecting the mixed solution to photo-crosslinking to obtain a hydrogel, and then freeze-drying to obtain the hemostatic sponge.

[0018] Preferably, in the mixed solution, the concentration of methacryloyl collagen is 5-15w / v%, the concentration of nanohydroxyapatite modified with epigallocatechin gallate is 0.1-2w / v%, so as to obtain a hemostatic sponge with better strength. Further preferably, in the mixed solution, the concentration of methacryloyl collagen is 5-15w / v%, the concentration of nanohydroxyapatite modified with epigallocatechin gallate is 0.5-1w / v%, and it has a good porosity. After freeze-drying, ColMA gel itself has a certain porosity. The introduction of an appropriate amount of HAP@EGCG can increase the tiny pores by providing more surface area, further improving the porosity of the hemostatic sponge, improving its ability to absorb liquid, and improving the hemostatic effect.

[0019] Preferably, the mixed solution is frozen at -70 to -90°C for 8 to 12 hours and then freeze-dried at -70 to -90°C for 40 to 50 hours.

[0020] The present invention also provides a use of the hemostatic sponge, specifically a drug for treating injuries, comprising the hemostatic sponge as described above, or the hemostatic sponge prepared by the preparation method described above. Preferably, the drug is a drug for treating oral injuries.

[0021] The hemostatic sponge of the present invention has the following beneficial effects:

[0022] (1) Prolonging the hemostatic effect: In the hemostatic sponge of the present invention, the combination of ColMA and HAP@EGCG can enhance the structural stability of the material, thereby providing a more lasting hemostatic effect, so that the hemostatic sponge can maintain an effective physical form in the high humidity and high enzyme environment of the oral cavity, thereby prolonging the duration of the effect.

[0023] (2) Enhanced antibacterial ability: EGCG in the hemostatic sponge has good antibacterial properties and can effectively inhibit the growth of common pathogens in the oral cavity, such as Porphyromonas gingivalis and Streptococcus. In addition, EGCG also has an anti-inflammatory effect. The present invention uses HAP@EGCG to provide a platform for releasing EGCG, which can achieve the sustained release of EGCG, provide continuous antibacterial and anti-inflammatory protection, and reduce the risk of infection.

[0024] (3) Improved biocompatibility: The natural properties of ColMA and HAP make the material more biocompatible and can significantly reduce the occurrence of local inflammation or allergic reactions compared to traditional synthetic materials.

[0025] In summary, the hemostatic sponge of the present invention, by combining ColMA with HAP@EGCG and optimizing the ratio, has good anti-collagenase degradation performance, good hemostatic, anti-inflammatory and antibacterial effects, can promote tissue regeneration and repair, and meet the multiple clinical needs for postoperative recovery. Patients are more comfortable when using the hemostatic sponge of the present invention, and the therapeutic effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a picture of a hemostatic sponge according to an embodiment of the present invention;

[0027] Figure 2 The results of the evaluation of the cytocompatibility of the hemostatic sponge according to the embodiment of the present invention are shown in FIG.

[0028] Figure 3 The figures are the evaluation results of the anti-inflammatory effect of the hemostatic sponge according to the embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] The methacrylylated type I collagen Col1MA and epigallocatechin gallate-modified nanohydroxyapatite HAP@EGCG used in the embodiments of the present invention are prepared by the following method.

[0032] (1) Preparation of Col1MA:

[0033] Weigh 5 g of type I collagen and dissolve it in 500 mL of deionized water, adjust the pH to 7, add 10 mL of methacrylic anhydride, and react at room temperature for 24 hours. The product, methacrylylated type I recombinant collagen (rColMA), is dialyzed in deionized water using a 3500 Da dialysis bag, centrifuged at 10,000 rpm for 15 minutes to remove impurities, and the supernatant is freeze-dried and stored at -20 °C until use.

[0034] (2) Preparation of HAP@EGCG:

[0035] 1 g of EGCG was dissolved in 50 mL of deionized water or a suitable solvent to prepare a 2% EGCG solution. 0.5 g of nanohydroxyapatite was added to the EGCG solution, ultrasonically dispersed for 15 min, and then stirred and mixed at 45 ° C to promote the interaction between EGCG and the surface of nanohydroxyapatite. After the reaction, it was washed with deionized water to remove unbound EGCG. The modified nanohydroxyapatite (HAP@EGCG) was freeze-dried for 48 h to obtain the final product.

[0036] Example 1

[0037] First, 0.05 g of Col1MA was dissolved in 1 mL of 0.2% (w / v) LAP solution, and 0.005 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0038] Example 2

[0039] First, 0.05 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.01 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0040] Example 3

[0041] First, 0.05 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.02 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate at a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0042] Example 4

[0043] First, 0.1 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.005 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete the cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0044] Example 5

[0045] First, 0.1 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.01 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0046] Example 6

[0047] First, 0.1 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.02 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete the cross-linking of the hydrogel. The Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0048] Example 7

[0049] First, 0.15 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.005 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0050] Example 8

[0051] First, 0.15 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.02 g of HAP@EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate at a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete the cross-linking of the hydrogel, and the Col1MA / HAP@EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried at -80°C for 48 hours to obtain the Col1MA / HAP@EGCG hemostatic sponge.

[0052] Comparative Example 1

[0053] First, 0.05 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate at a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking, and the Col1MA hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried for 48 hours to obtain the Col1MA hemostatic sponge.

[0054] Comparative Example 2

[0055] First, 0.15 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, vortexed for 30 seconds, and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles; then, it was irradiated with 365 nm ultraviolet light for 30 seconds to complete the cross-linking of the hydrogel, and the Col1MA hydrogel was obtained by demolding; finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried for 48 hours to obtain the Col1MA hemostatic sponge.

[0056] Comparative Example 3

[0057] First, 0.15 g of Col1MA was dissolved in 1 mL of 0.2% LAP solution, and 0.02 g of EGCG was added to the Col1MA solution. The mixture was vortexed for 30 seconds and then sonicated for 5 minutes. The two were mixed evenly and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the plate was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking of the hydrogel, and the Col1MA / EGCG hydrogel was obtained by demolding. Finally, the hydrogel was placed in a -80°C refrigerator overnight, and then freeze-dried for 48 hours to obtain the Col1MA / EGCG hemostatic sponge.

[0058] Comparative Example 4: Absorbent gelatin sponge produced by Jiangxi Xiangen Medical Technology Development Co., Ltd. (specification: Type B 20mm×20mm×50mm).

[0059] Figure 1 The pictures of the hemostatic sponges obtained in the above-mentioned Examples 1-8 are shown. The components of the hemostatic sponge samples of the above-mentioned Examples and Comparative Examples are shown in Table 1.

[0060] Table 1 Composition of hemostatic sponges of different embodiments and comparative examples

[0061]

[0062] Performance Test:

[0063] 1. Degradation performance

[0064] Test method: First weigh the hemostatic sponge and record the initial weight of the hemostatic sponge in different groups (the gelatin sponge is consistent with its mass), recorded as W0. Place the prepared hemostatic sponge sample and gelatin sponge in a 24-well plate, add a fixed volume of collagenase solution with a pipette, immerse the sample completely, and place it in a 37°C constant temperature shaker. After 12 hours, take out the samples, discard the excess degradation solution, freeze-dry, weigh, and record the weight, recorded as Wt. The degradation rate is calculated according to the following formula:

[0065]

[0066] Table 2 Degradation rate

[0067] Grouping Degradation rate Example 1 60.31±2.38％ Example 2 59.18±4.09％ Example 3 56.42±3.25％ Example 4 57.67±2.53％ Example 5 53.91±1.74％ Example 6 50.62±3.88％ Example 7 56.23±2.14％ Example 8 48.16±1.87％ Comparative Example 1 75.19±2.15％ Comparative Example 2 59.30±2.08％ Comparative Example 3 57.96±3.46％ Comparative Example 4 61.19±1.78％

[0068] Results: As can be seen from Table 2, as the concentration of Col1MA increases, the anti-degradation performance improves. When the concentration of Col1MA is between 10-15%, the degradation performance is significantly improved compared with existing commercial products. At the same time, the amount of HAP@EGCG added also affects the anti-degradation performance. In the range of 0.5%-2%, the more the amount added, the better the anti-degradation performance. Among them, the degradation rate of the hemostatic sponge of Example 8 in the collagenase degradation solution is 48.16±1.87% at 12h, while the gelatin sponge of Comparative Example 4 is 61.19±1.78%, indicating that the hemostatic sponge of the present invention has good stability and anti-degradation performance, and is more suitable for the oral environment.

[0069] 2. Porosity test

[0070] Test method: The porosity of the sponge is tested by the ethanol displacement method. Take a sponge with a radius of 5 mm and a height of 5 mm, weigh it and record it as M0, and fully immerse the sponge in the ethanol solution to make the sponge pores

[0071] The pores of the sponge are fully filled with ethanol, and the weight is taken out and weighed again, recorded as M. The calculation formula of sponge pores is as follows:

[0072]

[0073] Where M is the mass of the sponge after it is filled with ethanol, M0 is the initial weight of the sponge, and ρ ethanol is the density of ethanol (785 kg / m 3 ), V is the volume of the sponge.

[0074] Table 3 Porosity

[0075] Grouping Porosity Example 1 78.2±1.6％ Example 2 81.5±0.8％ Example 3 79.4±1.6％ Example 4 84.6±2.2％ Example 5 87.3±2.3％ Example 6 85.7±1.5％ Example 7 88.3±1.7％ Example 8 84.2±1.3％ Comparative Example 1 74.9±0.9％ Comparative Example 2 87.3±1.5％ Comparative Example 3 87.9±0.8％

[0076] The results show that the porosity of the sponge is a key factor in determining its ability to absorb liquid. A higher porosity can effectively help the sponge absorb exudate and achieve rapid hemostasis. As can be seen from Table 3, as the content of Col1MA increases, the porosity gradually increases, but the addition of HAP@EGCG can also increase the micropores by providing more surface area, thereby optimizing the porosity. However, too much HAP@EGCG may partially block the pores in the sponge, thereby reducing the porosity.

[0077] 3. Young's modulus

[0078] Test method: Place the sample on the sample table, take a sponge with a radius of 5mm and a height of 5mm, and adjust the height of the sample table so that both the upper and lower surfaces are in contact with the fixture. Compress at a constant rate of 0.05mm / min, and calculate the Young's modulus using the following formula:

[0079]

[0080] Where F is the compression force on the sponge, A is the cross-sectional area, L0 is the initial height of the sponge before compression, and ΔL is the change in the sponge height.

[0081] Table 4 Compression strength

[0082] Grouping Compression strength(kPa) Example 1 79.5±5.8 Example 2 82.4±3.2 Example 3 87.7±2.9 Example 4 101.5±4.6 Example 5 107.2±4.1 Example 6 111.3±2.3 Example 7 120.5±2.8 Example 8 134.2±4.3 Comparative Example 1 69.9±3.4 Comparative Example 2 110.6±2.7 Comparative Example 3 111.4±3.8

[0083] Test results: The compression strength of the sponge at 80% deformation is shown in Table 4. As the content of Col1MA increases, the mechanical strength of the sponge gradually increases. In addition, the amount of HAP@EGCG added also plays an important role in improving the mechanical strength of the sponge. The more the amount added, the higher the strength. This effect can also be clearly seen from the results of Example 7, Example 8 and Comparative Example 2. This may be because the addition of HAP@EGCG can make the three-dimensional network structure inside the sponge more compact and stable, which is beneficial for the sponge to resist large deformation.

[0084] 4. Antioxidant capacity test

[0085] Test method: The antioxidant performance of different embodiments and comparative examples was evaluated by scavenging 1,1-diphenyl-2-pyridylhydrazide (DPPH) free radicals. The mixture of different embodiments and comparative examples and DPPH reagent was stirred and incubated in the dark for 30 minutes, and the remaining DPPH was analyzed by UV-visible spectroscopy. The scavenging rate of DPPH was determined as follows:

[0086] D(%)=[[A 空白 -(A 测定 -A 对照 )] / A 空白 ]×100%

[0087] Table 5 Antioxidant capacity analysis

[0088]

[0089] Results: As shown in Table 5, the efficiency of scavenging free radicals increased with the increase of HAP@EGCG concentration, indicating that HAP@EGCG can effectively scavenge free radicals and has a better antioxidant effect.

[0090] 5. In vitro coagulation test

[0091] Test method: The in vitro whole blood coagulation index was used to study the coagulation-promoting effect of the hemostatic material. 100 μL of hydrogel was placed in a clean EP tube. 100 μL of anticoagulated blood and 20 μL of CaCl2 (0.2 mol / L) solution were added in sequence and mixed evenly. Then, the coagulation degree of the blood in the centrifuge tube was observed at 37°C. The test results are shown in Table 6.

[0092] Table 6 Whole blood coagulation index

[0093]

[0094]

[0095] The results show that the addition of ColMA has a significant coagulation effect, and the coagulation effect is enhanced with the increase of the added amount. At the same time, the content of HAP@EGCG also has a great influence on the coagulation index. This may be because a higher content of HAP@EGCG can form a denser and more stable three-dimensional network structure, which helps to capture blood cells, platelets and coagulation factors, promote their aggregation and stability, and thus accelerate the occurrence of coagulation. In addition, it can be seen that EGCG that is not combined with HAP has little effect on the coagulation effect, indicating that HAP in HAP@EGCG plays a major role in stabilizing the three-dimensional network structure.

[0096] 6. In vitro antibacterial test

[0097] Test method: After Porphyromonas gingivalis, Escherichia coli and Staphylococcus aureus were revived and cultured to the logarithmic growth phase, centrifuged, and the bacteria were collected and resuspended in saline. The final bacterial concentration was 1×108CFU / mL. After adding 500μL of the corresponding hydrogel solution to a 24-well plate, UV light was used to cure the gel. 500μL of saline was added to the control group. Subsequently, 100μL of the diluted bacterial suspension and 1mL of sterile saline were added to each well, incubated at 37°C for 24 hours, and then spread on BA medium or LB agar plates after gradient dilution and counted. The test results are shown in Table 7.

[0098] Table 7 In vitro antibacterial rate (%)

[0099]

[0100]

[0101] The results show that HAP@EGCG is the main antibacterial component, and the higher the addition amount, the stronger the antibacterial effect. Col1MA is a protein and bioactive factor, which has no effect on killing bacteria.

[0102] 7. Cytocompatibility evaluation

[0103] Test method: Sterilize the hemostatic sponge with ultraviolet light for 24 hours in advance. Then, extract the hemostatic sponge with DMEM complete medium at a mass ratio of 1:10 at 37°C for 24 hours to obtain the extract for cell culture in subsequent experiments.

[0104] L929 was digested with 0.05% trypsin, resuspended in DMEM, and inoculated in a 96-well plate at a density of 5000 cells per well. The cells were cultured in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 24 hours until the cells adhered to the wall. The culture medium was aspirated and washed with PBS, and the extract was added according to the corresponding grouping and continued to be cultured for 24 hours. After the culture was completed, the culture medium was aspirated and washed 3 times with PBS, and 100 μL CCK-8 working solution (DMEM basal culture medium containing 10% CCK-8) was added to each well. After incubation in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 30 minutes, the absorbance (OD) was measured at a wavelength of 450nm using an enzyme reader, and the cell survival rate was calculated according to the formula:

[0105]

[0106] Result description: The test results are as follows: Figure 2 As shown, the hemostatic sponges had no toxicity to cells, and the cell viability was higher than 80%, or even exceeded 100%, indicating that the prepared sponge materials had no obvious toxicity to cells and could promote cell proliferation to a certain extent.

[0107] 8. Anti-inflammatory evaluation

[0108] Test method: After the rats were anesthetized, the unilateral or bilateral maxillary or mandibular first molars of the rats were extracted with sterilized forceps or tooth extraction instruments. After tooth extraction, the wound usually had some bleeding. At this time, the hemostatic sponge to be tested was placed on the wound. After the sponge was implanted, the wound was closed with 7-0 absorbable sutures. The periodontal tissue samples were taken 7 days after surgery, washed in pre-cooled PBS (0.02mol / L, pH 7.0-7.2) to remove blood, and weighed for later use; 1.0g of tissue blocks were transferred into a glass homogenizer, and 5mL of pre-cooled PBS was added for full grinding. This process should be carried out on ice; the obtained homogenate was repeatedly frozen and thawed twice, and the prepared homogenate was centrifuged at 5000×g for 5 minutes, and the supernatant was collected for detection using an ELISA kit.

[0109] Result description: The test results are as follows: Figure 3 As shown, compared with the saline group and the commercially available material group, the ColMA sponge loaded with HAP@EGCG can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β, providing a stable microenvironment for periodontal tissue.

[0110] In summary, it can be seen that the hemostatic sponge of the embodiment of the present invention combines methacrylylated collagen with hydroxyapatite to improve the porosity of the material and enhance the hemostatic performance; at the same time, it can also improve the strength and material stability, and can better adapt to high humidity and high enzyme environments. Moreover, the hydroxyapatite is nano-hydroxyapatite HAP@EGCG modified by EGCG. HAP provides a sustained-release platform for EGCG, which can inhibit bacteria for a long time, control postoperative inflammation, significantly reduce the risk of infection, and promote tissue regeneration. In addition, the combination with HAP also enhances the healing effect and meets multiple postoperative needs.

[0111] The hemostatic sponge of the embodiment of the present invention has good anti-collagenase degradation, hemostasis, antibacterial, anti-inflammatory and healing promotion effects, and has good biocompatibility, and is particularly suitable for complex oral environments, ensuring that it still maintains good performance under the action of humidity and enzymes.

[0112] The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A multifunctional hemostatic sponge, characterized in that: The hemostatic sponge comprises a matrix formed by cross-linking methacryloyl collagen and nano-hydroxyapatite modified with epigallocatechin gallate, and the mass ratio of the methacryloyl collagen to the nano-hydroxyapatite modified with epigallocatechin gallate is 2-30:

1.

2. The hemostatic sponge according to claim 1, characterized in that: The epigallocatechin gallate modified nano hydroxyapatite is obtained by adding nano hydroxyapatite into the epigallocatechin gallate solution, dispersing, stirring, mixing, reacting at 40-50°C, and then washing and drying.

3. The hemostatic sponge according to claim 2, characterized in that: The concentration of the epigallocatechin gallate solution is 1-10 w / v%, and the mass ratio is epigallocatechin gallate: nano-hydroxyapatite = 0.2-0.5:

1.

4. The hemostatic sponge according to claim 1, characterized in that: The methacryloyl collagen is methacryloyl type I collagen.

5. The hemostatic sponge according to claim 4, characterized in that: The methacryloyl collagen is prepared by dropping methacrylic anhydride into a type I collagen aqueous solution with a pH of 7, and reacting at room temperature. The collagen: water: methacrylic anhydride = 5 g: 500 mL: 10 mL.

6. The method for preparing the hemostatic sponge according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: adding nano hydroxyapatite modified with epigallocatechin gallate to a methacrylylated collagen solution containing a photoinitiator, mixing the mixture evenly to obtain a mixed solution, then subjecting the mixed solution to photo-crosslinking molding to obtain a hydrogel, and freeze-drying the hydrogel to obtain the hemostatic sponge.

7. The preparation method according to claim 6, characterized in that: In the mixed solution, the concentration of methacrylylated collagen is 5-15 w / v%, and the concentration of nano-hydroxyapatite modified with epigallocatechin gallate is 0.1-2 w / v%.

8. The preparation method according to claim 6, characterized in that: The mixed solution is frozen at -70 to -90°C for 8 to 12 hours, and then freeze-dried at -70 to -90°C for 40 to 50 hours.

9. A drug for treating injury, characterized in that: The invention comprises the hemostatic sponge according to any one of claims 1 to 5, or the hemostatic sponge prepared by the preparation method according to any one of claims 6 to 8.

10. The drug according to claim, characterized in that The medicine is a medicine for treating oral injuries.

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