A multifunctional hemostatic sponge and its preparation method and application

Abstract

The present invention relates to a multifunctional hemostatic sponge, its preparation method, and application. The sponge comprises a matrix formed by cross-linking methacryloylated collagen and epigallocatechin gallate-modified nanohydroxyapatite, with the mass ratio of methacryloylated collagen to epigallocatechin gallate-modified nanohydroxyapatite being 2 to 30:1. By combining ColMA with HAP@EGCG and optimizing the ratio, the resulting hemostatic sponge exhibits excellent resistance to collagenase degradation, as well as hemostatic, anti-inflammatory, and antibacterial effects. It can promote tissue regeneration and repair, provide greater patient comfort, and enhance therapeutic efficacy.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, 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, bacterial, and rich in enzymes, traditional hemostatic materials have significant deficiencies in hemostasis, antibacterial and anti-inflammatory properties, and healing-promoting functions. Furthermore, the complex structure of the oral cavity makes it difficult to reach hidden wound sites 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, oral hemostatic materials 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-promoting effects: Inflammatory reactions after surgery are relatively common, and existing materials mostly focus on hemostasis and lack the multifunctionality of anti-inflammatory and tissue healing promotion.

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

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

[0009] In view of the above-mentioned deficiencies in the 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 rapid hemostasis and antibacterial and anti-inflammatory functions.

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

[0011] Hydroxyapatite (HAP) has a hemostatic effect, and EGCG (epigallocatechin gallate) has excellent antibacterial properties, helping to control inflammatory responses. The present invention cross-links methacryloyl-collagen (ColMA) with EGCG-modified HAP, creating a sustained-release platform for EGCG. The three components work together to support each other, resulting in a hemostatic material with excellent biocompatibility, structural stability, increased strength, and improved hemostatic properties. It also exhibits excellent anti-inflammatory and antibacterial effects, promoting tissue regeneration and repair, and promoting healing. In particular, it can maintain an effective physical form for a long time in the high-humidity and high-enzyme environment of the oral cavity, resulting in a long-lasting effect, greater patient comfort, and improved therapeutic effects.

[0012] Preferably, the epigallocatechin gallate-modified nanohydroxyapatite is obtained by adding nanohydroxyapatite to the epigallocatechin gallate solution, dispersing the nanohydroxyapatite, stirring, mixing, reacting at 40-50° C., washing, and drying. The epigallocatechin gallate solution can be prepared by dissolving epigallocatechin gallate in deionized water or a suitable solvent (e.g., aqueous ethanol). Drying can be performed by heat drying or freeze drying, with freeze drying being preferred for better dispersibility.

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

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

[0015] Further preferably, the methacrylated collagen is prepared by dropwise adding methacrylic anhydride to a type I collagen aqueous solution with a pH of 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 nanohydroxyapatite modified with epigallocatechin gallate to a methacryloyl collagen solution containing a photoinitiator, mixing uniformly 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 5-15 w / v%, the concentration of epigallocatechin gallate-modified nanohydroxyapatite is 0.1-2 w / v%, so as to obtain a hemostatic sponge with better strength. Further preferably, in the mixed solution, the concentration of methacryloyl collagen is 5-15 w / v%, the concentration of epigallocatechin gallate-modified nanohydroxyapatite is 0.5-1 w / v%, and the porosity is better. After freeze-drying, ColMA gel itself has a certain porosity. The appropriate amount of HAP@EGCG introduced can increase the tiny pores by providing more surface area, further improving the porosity of the hemostatic sponge, enhancing 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 above preparation method. 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) Prolonged 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, allowing the hemostatic sponge to 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 oral pathogens such as Porphyromonas gingivalis and Streptococcus. In addition, EGCG also has anti-inflammatory effects. The present invention uses HAP@EGCG to provide a platform for releasing EGCG, which can achieve 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, obtained by combining ColMA and HAP@EGCG in an optimized ratio, has excellent resistance to collagenase degradation, as well as good hemostatic, anti-inflammatory and antibacterial effects. It can promote tissue regeneration and repair, meeting the multiple clinical needs for postoperative recovery. Patients feel more comfortable when using the hemostatic sponge of the present invention, and the treatment 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 cytocompatibility evaluation 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, which, however, are not intended to limit the present invention in any way. 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 were commercially available.

[0031] The methacrylylated type I collagen Col1MA and epigallocatechin gallate-modified nanohydroxyapatite HAP@EGCG used in the examples of the present invention were 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, then add 10 mL of methacrylic anhydride dropwise. Allow to react at room temperature for 24 hours. The resulting methacryloylated recombinant type I collagen (rColMA) was dialyzed against deionized water using a 3500 Da dialysis bag. Impurities were removed by centrifugation at 10,000 rpm for 15 minutes. The supernatant was lyophilized and stored at -20°C until use.

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

[0035] Dissolve 1g of EGCG in 50mL of deionized water or a suitable solvent to prepare a 2% EGCG solution. Add 0.5g of nanohydroxyapatite to the EGCG solution and ultrasonically disperse for 15 minutes. Then, stir and mix at 45°C to promote interaction between EGCG and the nanohydroxyapatite surface. After the reaction, wash with deionized water to remove unbound EGCG. The modified nanohydroxyapatite (HAP@EGCG) was freeze-dried for 48 hours 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. 0.005 g of HAP@EGCG was added to the Col1MA solution, 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 hydrogel was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.01 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.02 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.005 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.01 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.02 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.005 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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. 0.02 g of HAP@EGCG was added to the Col1MA solution, vortexed for 30 seconds, and then sonicated for 5 minutes. The mixture was evenly mixed and placed in a 48-well plate under a vacuum of -1 bar to remove bubbles. Then, the hydrogel was irradiated with 365 nm UV light for 30 seconds to complete cross-linking. The Col1MA / HAP@EGCG hydrogel was demolded to obtain the Col1MA / HAP@EGCG hydrogel. 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 under 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 demolding to obtain the Col1MA hydrogel. 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, the hydrogel was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking, and demolded to obtain the Col1MA hydrogel. 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. 0.02 g of EGCG was added to the Col1MA solution, 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 hydrogel was irradiated with 365 nm ultraviolet light for 30 seconds to complete cross-linking. The Col1MA / EGCG hydrogel was demolded to obtain the Col1MA / EGCG hydrogel. 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. (specifications: Type B 20 mm × 20 mm × 50 mm).

[0059] Figure 1 The pictures of the hemostatic sponges obtained in the above Examples 1-8 are shown. The components of the hemostatic sponge samples of the above 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 testing:

[0063] 1. Degradation performance

[0064] Test method: First, weigh the hemostatic sponge and record the initial weight of the different groups of hemostatic sponges (the gelatin sponge should be 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 using a pipette to completely immerse the sample, and place it in a 37°C constant temperature shaker. After 12 hours, remove the sample, discard the excess degradation solution, lyophilize, 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 shown in Table 2, degradation resistance improves with increasing Col1MA concentration. At Col1MA concentrations between 10% and 15%, degradation performance is significantly improved compared to existing commercially available products. The amount of HAP@EGCG added also affects degradation resistance. Within the range of 0.5% to 2%, the higher the HAP@EGCG addition, the better the degradation resistance. The degradation rate of the hemostatic sponge in Example 8 in the collagenase degradation solution was 48.16 ± 1.87% after 12 hours, compared to 61.19 ± 1.78% for the gelatin sponge in Comparative Example 4. This demonstrates that the hemostatic sponge of the present invention exhibits superior stability and degradation resistance, making it more suitable for oral environments.

[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 5mm and a height of 5mm, weigh it and record it as M0, and fully immerse the sponge in ethanol solution to make the sponge pores

[0071] The pores of the sponge are fully filled with ethanol, then the pores are taken out and weighed again, which is 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 indicate that the porosity of the sponge is a key factor in determining its ability to absorb fluids. A higher porosity effectively helps the sponge absorb exudate and achieve rapid hemostasis. As shown in Table 3, the porosity increases with increasing Col1MA content. However, the addition of HAP@EGCG can also optimize the porosity by increasing the surface area and increasing the micropores. However, excessive HAP@EGCG may partially block the pores in the sponge, 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. The Young's modulus is calculated using the following formula:

[0079]

[0080] Where F is the compressive 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 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 compressive strength of the sponge at 80% deformation is shown in Table 4. As the Col1MA content increases, the mechanical strength of the sponge gradually increases. Furthermore, the addition of HAP@EGCG also plays a significant role in improving the mechanical strength of the sponge: a higher addition increases the strength. This effect is also clearly evident in the results of Examples 7 and 8, as well as Comparative Example 2. This is likely because the addition of HAP@EGCG makes the three-dimensional network structure within the sponge denser and more stable, helping the sponge resist large deformations.

[0084] 4. Antioxidant capacity test

[0085] Test Method: The antioxidant properties of different examples and comparative examples were evaluated using a method for scavenging 1,1-diphenyl-2-pyridylhydrazine (DPPH) free radicals. A mixture of different examples and comparative examples with a DPPH reagent was incubated in the dark with stirring for 30 minutes, and the remaining DPPH was analyzed using UV-visible spectroscopy. The formula for determining the DPPH scavenging rate is:

[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 better antioxidant effect.

[0090] 5. In vitro coagulation test

[0091] Test Method: The coagulation-promoting effect of the hemostatic material was studied using the in vitro whole blood coagulation index. 100 μL of the hydrogel was placed in a clean EP tube. 100 μL of anticoagulated blood and 20 μL of a 0.2 mol / L CaCl₂ solution were then added and mixed thoroughly. The degree of coagulation of the blood in the centrifuge tube was then 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, which is enhanced with the increase in the added amount. At the same time, the content of HAP@EGCG also has a significant impact 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. This structure helps to capture blood cells, platelets and coagulation factors, promote their aggregation and stabilization, and thus accelerate the occurrence of coagulation. In addition, it can be seen that EGCG not bound to 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: Porphyromonas gingivalis, Escherichia coli, and Staphylococcus aureus were revived and cultured to the logarithmic growth phase. The bacteria were centrifuged, collected, and resuspended in saline to a final bacterial concentration of 1×108 CFU / mL. 500μL of the corresponding hydrogel solution was added to a 24-well plate and cured under UV light to form a 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. The plates were incubated at 37°C for 24 hours, and after gradient dilution, the plates were spread on BA medium or LB agar plates 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 that has no effect on killing bacteria.

[0102] 7. Cytocompatibility evaluation

[0103] Testing Method: Sterilize the hemostatic sponge by UV irradiation for 24 hours. Then, soak the sponge in DMEM complete medium at a mass ratio of 1:10 at 37°C for 24 hours. The extract is used for cell culture in subsequent experiments.

[0104] L929 was digested with 0.05% trypsin, resuspended in DMEM, and seeded 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. The culture medium was discarded, washed with PBS, and the extract was added according to the corresponding group and cultured for 24 hours. After the culture was completed, the culture medium was discarded, washed 3 times with PBS, and 100 μL CCK-8 working solution (DMEM basal medium containing 10% CCK-8) was added to each well. After incubation for 30 minutes in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2), the absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader, and the cell viability 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 anesthesia, rats were anesthetized and their maxillary or mandibular first molars were extracted unilaterally or bilaterally using sterile forceps or dental extraction instruments. Following tooth extraction, the wound typically bleeds slightly. A hemostatic sponge to be tested was placed on the wound. After the sponge was implanted, the wound was closed with 7-0 absorbable sutures. Seven days after surgery, periodontal tissue samples were collected, cleaned in pre-chilled PBS (0.02 mol / L, pH 7.0-7.2) to remove blood, and weighed for later use. A 1.0 g tissue block was transferred to a glass homogenizer and thoroughly ground with 5 mL of pre-chilled PBS. This process was performed on ice. The resulting homogenate was repeatedly frozen and thawed twice. The prepared homogenate was centrifuged at 5000 × g for 5 minutes, and the supernatant was collected for analysis using an ELISA kit.

[0109] Result description: The test results are as follows: Figure 3 As shown in the results, compared with the normal saline group and the commercial 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 methacrylated collagen with hydroxyapatite to increase 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. In addition, the hydroxyapatite is nanohydroxyapatite HAP@EGCG modified with EGCG. HAP provides a sustained-release platform for EGCG, which can lastingly inhibit bacteria, 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. It is particularly suitable for complex oral environments and ensures that it still maintains good performance under the action of humidity and enzymes.

[0112] The technical features of the above-described embodiments can be combined in any combination. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are 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, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of this invention shall be based on the attached claims.

Claims

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

1. The epigallocatechin gallate-modified nanohydroxyapatite is obtained by adding nanohydroxyapatite into an epigallocatechin gallate solution, dispersing, stirring, mixing, reacting at 40-50° C., and then washing and drying. 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.

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

3. The hemostatic sponge according to claim 2, characterized in that The methacrylated collagen is prepared by dropwise adding methacrylic anhydride to 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.

4. The method for preparing the hemostatic sponge according to any one of claims 1 to 3, wherein: The method comprises the following steps: adding nano-hydroxyapatite modified with epigallocatechin gallate to a methacryloyl collagen solution containing a photoinitiator, mixing the mixture evenly to obtain a mixed solution, then subjecting the mixed solution to photo-crosslinking to form a hydrogel, and freeze-drying the hydrogel to obtain the hemostatic sponge.

5. The preparation method according to claim 4, characterized in that In the mixed solution, the concentration of methacryloyl collagen is 5-15 w / v%, and the concentration of epigallocatechin gallate-modified nanohydroxyapatite is 0.1-2 w / v%.

6. The preparation method according to claim 4, 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.

7. A drug for treating injury, characterized in that: The invention comprises the hemostatic sponge according to any one of claims 1 to 3, or the hemostatic sponge prepared by the preparation method according to any one of claims 4 to 6.

8. The drug according to claim 7, characterized in that The medicine is a medicine for treating oral injuries.

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