Bioactive glass composite antibacterial hemostatic frozen gel sponge as well as preparation method and application thereof

Abstract

The invention discloses bioactive glass composite antibacterial hemostatic frozen gel sponge as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: adding EDC and NHS into an SA aqueous solution under continuous stirring, continuously stirring, adding ZnMBG into a CCS aqueous solution, uniformly dispersing, mixing and stirring the obtained mixed solution, pouring the mixed solution into a mold, standing at low temperature, carrying out primary crosslinking and freeze drying, soaking the obtained frozen gel into an anhydrous calcium chloride solution, carrying out secondary crosslinking, and carrying out freeze drying to obtain the hydrogel. And freeze-drying to obtain the product. The obtained sponge has a macroporous structure, can quickly absorb blood and form a physical barrier, activates a blood coagulation pathway by releasing Ca < 2 + > and Zn < 2 + >, and promotes red blood cell aggregation and blood clot formation; zn < 2 + > and positive groups of CCS are synergistically antibacterial, so that infection is prevented; the material has shape memory ability and biocompatibility, and is suitable for efficient hemostasis and infection control of incompressible wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and specifically relates to a bioactive glass composite antibacterial hemostatic cryogel sponge, a preparation method thereof, and an application thereof. Background Art

[0002] Bleeding is one of the most common injuries at the scene of various accidents. Compared with traditional wounds, non-compressible and irregular wounds such as ballistic wounds and penetrating wounds have the characteristics of deeper wound sites, large amounts of wound bleeding, high requirements for hemostasis speed, and easy occurrence of infections. If timely hemostasis cannot be achieved, it will endanger the life of the patient. Currently, various hemostatic devices, topical hemostatic agents, surgical sealants, adhesives, and dressings sold on the market are applicable to bleeding hemostasis at the surface of the body, shallow wound surfaces, and compressible wound surfaces, but are not applicable to hemostasis of the head, internal organs, arteriovenous ruptures, and deep and narrow non-compressible wound surfaces with irregular shapes. In addition, since blood is a natural medium for bacteria, once the human body has an open wound, it is easy to cause wound infection. Current commercial hemostatic materials have been proven to be able to effectively shorten the hemostasis time, but it is difficult for them to deal with complex non-compressible wounds, and the hemostasis mechanism is relatively single, only having the ability to stop bleeding, and cannot protect the tissue wound surface from being infected after hemostasis.

[0003] The inherent macroporous structure of the cryogel ensures the flow of water and endows the cryogel with excellent water absorption capacity, capable of absorbing a large amount of blood from the wound area and promoting the hemostasis process. The pore structure of the cryogel is very similar to the structure of the extracellular matrix, and can also effectively remove wound exudate and keep the wound area moist. These characteristics make the cryogel have good application prospects in the field of hemostasis. However, the cryogel usually exhibits poor mechanical properties. Without reasonable modification, when applied to the bleeding site, it cannot form a strong physical barrier and cannot cope with the impact of blood flow, thus unable to achieve hemostasis of non-compressible wounds.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a bioactive glass composite antibacterial hemostatic cryogel sponge with good mechanical properties and capable of coping with non-compressible wound bleeding through multiple hemostasis mechanisms, a preparation method thereof, and an application thereof. Summary of the Invention

[0005] In view of this, the present invention provides a bioactive glass composite antibacterial hemostatic cryogel sponge, a preparation method thereof, and an application thereof. The cryogel prepared by forming a multi-crosslinked network through the interaction of amide bonds, ionic bonds, and hydrogen bonds has rapid water absorption and good mechanical properties, can still rapidly absorb water and restore its shape under large compression deformation conditions, concentrate coagulation active factors, and form a strong physical barrier at the bleeding site, capable of quickly completing hemostasis for non-compressible wound bleeding. The negatively charged zinc-doped mesoporous bioactive glass nanoparticles in the sponge can release Ca2+ , Zn 2+ and other bioactive ions can activate the endogenous coagulation pathway and the exogenous coagulation pathway, promote the aggregation of red blood cells and platelets at the same time, accelerate the formation of blood clots, and can also effectively kill bacteria on the wound surface to avoid wound infection. The cryogel sponge has both good biocompatibility and biodegradability, and has great clinical application potential in the field of hemostasis for incompressible wounds.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation method of a bioactive glass composite antibacterial hemostatic cryogel sponge, comprising the following steps:

[0008] (1) Dissolve sodium alginate (SA) in deionized water to obtain an aqueous sodium alginate solution. Add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution under continuous stirring and continue stirring to obtain a mixed solution 1;

[0009] (2) Dissolve carboxylated chitosan (CCS) in deionized water to obtain an aqueous carboxylated chitosan solution. Add zinc-doped mesoporous bioactive glass (ZnMBG) to the aqueous carboxylated chitosan solution and disperse evenly to obtain a mixed solution 2;

[0010] (3) Mix and stir the mixed solution 1 and the mixed solution 2, pour them into a mold, and let them stand at low temperature for preliminary cross-linking, and then freeze-dry to obtain a cryogel with a macroporous structure;

[0011] (4) Immerse the obtained cryogel in an anhydrous calcium chloride solution for secondary cross-linking, and then freeze-dry to obtain the above-mentioned bioactive glass composite antibacterial hemostatic cryogel sponge with shape memory function.

[0012] The beneficial effects of the present invention:

[0013] 1. The raw materials selected in the present invention and the obtained cryogel sponge have good biocompatibility, the obtained product is non-toxic, has good cell compatibility, its in vitro hemolysis rate <5%, and there is no significant inhibition of the proliferation of L929 fibroblasts.

[0014] 2. The cryogel sponge obtained in the present invention can quickly absorb water and has good mechanical properties, has shape memory ability, can absorb water and restore its shape after >90% compressive deformation, and the structure is complete, and can form a physical barrier on the incompressible wound surface to complete hemostasis.

[0015] 3. The cryogel sponge obtained in the present invention can promote Ca 2+ and Zn 2Enter the wound, activate the coagulation cascade reaction and accelerate the hemostasis process.

[0016] 4. The present invention introduces zinc-doped bioactive glass, whose mesoporous structure can concentrate blood, accelerate thrombus formation, and release Ca 2+ and Zn 2+ . Combining with its negatively charged surface, it activates the intrinsic and extrinsic coagulation pathways to promote the formation of blood clots, thereby achieving rapid hemostasis.

[0017] 5. The carboxylated chitosan molecular chain in the present invention has positively charged groups, which together with the Zn 2+ released by bioactive glass achieve wound surface antibacterial and prevent wound surface infection, and its bactericidal rate against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) is ≥90%.

[0018] 6. The cryogel sponge is expected to become an excellent hemostatic material with great clinical application potential.

[0019] The invention principle of the present invention: First, prepare an aqueous solution of carboxylated chitosan, that is, an aqueous solution of CCS. Using the sol-gel method and ion doping method, combined with the sacrificial template method, prepare mesoporous zinc-doped bioactive glass nanoparticles, that is, ZnMBG. Disperse ZnMBG in the aqueous solution of CCS, stir and mix evenly to obtain a CCS composite solution. Then prepare an aqueous solution of sodium alginate, that is, an aqueous solution of SA, add an EDC / NHS crosslinking agent to activate the carboxyl groups on the main chain of SA. After sufficient activation, mix and stir the CCS composite solution and the SA solution to carry out a crosslinking reaction. Among them, the activated carboxyl groups on the main chain of SA will form amide bonds with the amino groups on CCS. At the same time, the Ca 2+ and Zn 2+ released by ZnMBG can form ionic bonds with the carboxyl groups on SA to form an "eggshell structure". This crosslinking reaction initially enhances the mechanical properties of the cryogel, and the cryogel sponge is obtained by freeze-drying. Then, soak it in a calcium chloride solution, and the unreacted carboxyl groups on the main chain of SA will further crosslink with Ca 2+ , thereby further enhancing the mechanical properties of the cryogel, and the cryogel sponge is obtained again after freeze-drying.

[0020] In the prior art, sodium alginate, chitosan and bioactive glass are directly mixed to obtain a hemostatic material. First, the chitosan used in the prior art can only be dissolved in an acid solution, while carboxylated chitosan has water solubility and can be directly dissolved in an aqueous solution.

[0021] 1. Mechanical properties:

[0022] Prior art: The hemostatic material obtained by direct mixing is through physical bonding such as electrostatic interaction, relying only on intermolecular forces (such as van der Waals forces). The material structure is loose, and the compression modulus is usually low, unable to withstand the blood flow impact of incompressible wounds.

[0023] This application: Dual cross-linked network: First is amide bond cross-linking. EDC / NHS activates the carboxyl group of sodium alginate (SA) to form a covalent bond with the amino group of carboxylated chitosan (CCS), constructing a rigid framework. During the mixing process, the zinc-doped bioactive glass releases calcium ions and zinc ions, which can initially perform ionic cross-linking, enabling the material to maintain a good morphology during subsequent ionic cross-linking processes; Second is cross-linking with Ca ions. When soaking in calcium chloride for the second time, the unreacted carboxyl groups of SA form an "eggshell structure" with Ca ions, enhancing the network toughness.

[0024] 2. Structural stability

[0025] Prior art: Physically mixed materials rapidly swell and disintegrate when encountering blood, unable to form a stable barrier, and the dressing needs to be frequently changed.

[0026] This application: The dual cross-linked network endows the material with good compression resistance and fatigue resistance (shape memory ability), can absorb water and swell, and form a stable physical barrier on the wound surface.

[0027] Furthermore, in step (1), the mass-volume ratio of sodium alginate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride, N-hydroxysuccinimide, and deionized water is (0.1 - 0.5) g : (0.05 - 0.25) g : (0.05 - 0.25) g : 10 mL.

[0028] Furthermore, in step (2), the mass-volume ratio of carboxylated chitosan, zinc-doped mesoporous bioactive glass, and deionized water is (0.1 - 0.5) g : (0.008 - 0.1) g : 10 mL.

[0029] Beneficial effects of adopting the above further technical solution: The concentration of the carboxylated chitosan solution can ensure the uniform dispersion of ZnMBG, avoid agglomeration, and at the same time maintain the fluidity of the solution, facilitating subsequent mixing with the sodium alginate solution.

[0030] Furthermore, in step (2), the above zinc-doped mesoporous bioactive glass is composed of the following components in molar percentages: SiO2 60%, P2O5 4%, CaO 31%, ZnO 5%, with a particle size of 100 - 400 nm and a mesoporous pore diameter of 2 - 15 nm.

[0031] Advantages of adopting the above further technical solution: The mesoporous structure with a size of 2 - 15 nm significantly increases the specific surface area, promotes the adsorption and concentration of blood coagulation factors, and accelerates thrombus formation; the particle size range of 100 - 400 nm ensures the uniform dispersion of particles in the cryogel, avoiding structural defects caused by local stress concentration.

[0032] Further, in step (3), the preparation method of the above zinc-doped mesoporous bioactive glass includes the following steps:

[0033] Dissolve cetyltrimethylammonium chloride (CTAB) in deionized water, slowly add ethyl acetate under continuous stirring and continue stirring, then slowly add ammonia water and continue stirring to obtain a mixture. Finally, add tetraethoxysilane (TEOS), triethoxyphosphate (TEP), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), and zinc nitrate hexahydrate (Zn(NO3)2·6H2O) to the obtained mixture at intervals for a period of time and continue stirring. Centrifuge to collect the product, and wash the precipitate with alternating water and ethanol multiple times. Dry the precipitate and calcine it to obtain zinc-doped mesoporous bioactive glass.

[0034] Advantages of adopting the above further technical solution: The present invention uses the sol-gel method and the ion doping method, in combination with the sacrificial template method, to prepare mesoporous zinc-doped bioactive glass nanoparticles.

[0035] Even further, in step (3), the preparation method of the above zinc-doped mesoporous bioactive glass includes the following steps:

[0036] Dissolve 2.1 g of cetyltrimethylammonium chloride in 99 mL of deionized water, slowly add 30 mL of ethyl acetate under continuous stirring and continue stirring for 30 min to form uniform microemulsion droplets, with a stirring speed of 200 - 400 rpm. Then slowly add 21 mL of ammonia water with a concentration of 2 mol / L and continue stirring for 15 min, with a stirring speed of 200 - 400 rpm, to obtain a mixture. Finally, add 12.4998 g of tetraethoxysilane, 1.4572 g of triethoxyphosphate, 7.3207 g of calcium nitrate tetrahydrate, and 1.4876 g of zinc nitrate hexahydrate to the obtained mixture at intervals of 30 min and continue stirring for 4 h, with a stirring speed of 200 - 400 rpm. Centrifuge to collect the product, with a centrifugation speed of 8000 revolutions per minute and a centrifugation time of 5 min. Wash the precipitate with alternating water and ethanol three times. Dry the precipitate in an oven at 60 °C for 24 h, and then calcine it at 650 °C for 3 h to obtain zinc-doped mesoporous bioactive glass.

[0037] Further, in step (3), the mass ratio of sodium alginate in mixed solution 1 to carboxylated chitosan in mixed solution 2 is 1:1.

[0038] Further, in step (3), it is left standing for 24 - 48 h at a temperature of -20 to -80°C, the temperature for freeze-drying is -20 to -80°C, the time for freeze-drying is 24 - 48 h, and the vacuum degree is 1 - 15 Pa.

[0039] Beneficial effects of adopting the above further technical solution: within the temperature range of -20 to -80°C, the cooling rate of the solution slows down relatively, water molecules have sufficient time to form ice crystals, and during the slow growth process of the ice crystals, adjacent ice crystals gradually fuse, ultimately forming a continuous macroporous structure, endowing the sponge with rapid liquid absorption ability.

[0040] Furthermore, in step (2), the zinc-doped mesoporous bioactive glass is added to the carboxylated chitosan aqueous solution and dispersed uniformly by ultrasonic treatment, magnetic stirring or a homogenizer.

[0041] Furthermore, in step (1), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride and N-hydroxysuccinimide are added to the sodium alginate aqueous solution under continuous stirring and then stirring is continued, the stirring speed is 500 - 800 rpm, and the stirring time is 10 - 30 min;

[0042] In step (2), the zinc-doped mesoporous bioactive glass is added to the carboxylated chitosan aqueous solution and stirred magnetically, the stirring speed is 200 - 400 rpm, and the stirring time is 10 - 30 min;

[0043] In step (3), the mixed solution 1 and the mixed solution 2 are mixed and stirred, the stirring speed is 200 - 400 rpm, and the stirring time is 0.5 - 1 h.

[0044] Further, in step (4), the mass concentration of the above calcium chloride solution is 0.01 - 0.05 g / mL, the soaking time is 0.5 - 1.5 h, the temperature for freeze-drying is -20 to -80°C, the time for freeze-drying is 24 - 48 h, and the vacuum degree is 1 - 15 Pa.

[0045] Beneficial effects of adopting the above further technical solution: crosslinking is carried out with a lower concentration of calcium chloride to avoid excessive local crosslinking caused by too high calcium ion concentration during the secondary crosslinking process, while maintaining the macroporous structure of the sponge and enhancing the mechanical strength.

[0046] The present invention also provides a bioactive glass composite antibacterial hemostatic cryogel sponge prepared by the above method.

[0047] The present invention also provides an application of the above bioactive glass composite antibacterial hemostatic cryogel sponge in the preparation of medical materials for incompressible wound hemostasis and infection control. Description of the Drawings

[0048] Figure 1Among them, Figure a is the scanning electron microscope result diagram of ZnMBG, and Figure b is the transmission electron microscope result diagram of ZnMBG.

[0049] Figure 2 Among them, Figure a is one of the scanning electron microscope result diagrams of SA / CCS-1 sponge (magnification: 50×), and Figure b is another scanning electron microscope result diagram of SA / CCS-1 sponge (magnification: 2000×).

[0050] Figure 3 Among them, Figure a is one of the scanning electron microscope result diagrams of SA / CCS / ZnMBG-1 sponge (magnification: 50×), and Figure b is another scanning electron microscope result diagram of SA / CCS / ZnMBG-1 sponge (magnification: 2000×).

[0051] Figure 4 Among them, Figure a and Figure b are the compressive stress-strain curve diagrams of the cryogel sponge, and Figure c is the compressive stress-strain cyclic curve diagram of the cryogel sponge.

[0052] Figure 5 It is the in vitro hemolysis test diagram of the cryogel sponge.

[0053] Figure 6 It is the in vitro coagulation test diagram of the cryogel sponge.

[0054] Figure 7 Among them, Figure a is the red blood cell adhesion test diagram of the cryogel sponge, and Figure b is the platelet adhesion test diagram of the cryogel sponge.

[0055] Figure 8 It is the L929 cell proliferation test diagram of the cryogel sponge.

[0056] Figure 9 It is the antibacterial rate test diagram of the cryogel sponge. Specific embodiments

[0057] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] Example 1

[0059] A preparation method of a bioactive glass composite antibacterial hemostatic cryogel sponge includes the following steps:

[0060] (1) Dissolve 2.1 g of cetyltrimethylammonium chloride in 99 mL of deionized water. While continuously stirring, slowly add 30 mL of ethyl acetate and continue stirring for 30 min to form uniform microemulsion droplets. The stirring speed is 400 rpm. Then, slowly add 21 mL of 2 mol / L ammonia water and continue stirring for 15 min with a stirring speed of 400 rpm to obtain a mixture. Finally, add 12.4998 g of tetraethoxysilane, 1.4572 g of triethoxyphosphate, 7.3207 g of calcium nitrate tetrahydrate, and 1.4876 g of zinc nitrate hexahydrate to the obtained mixture at intervals of 30 min and continue stirring for 4 h with a stirring speed of 400 rpm. Centrifuge to collect the product at a centrifugation speed of 8000 revolutions per minute for 5 min. Wash the precipitate three times with alternating water and ethanol. Dry the precipitate in an oven at 60 °C for 24 h, and then calcine it at 650 °C for 3 h to obtain zinc-doped mesoporous bioactive glass. The zinc-doped mesoporous bioactive glass is composed of the following components in molar percentages: SiO₂ 60%, P₂O₅ 4%, CaO 31%, ZnO 5%, with a particle size of 100 - 400 nm and a mesoporous pore size of 2 - 15 nm;

[0061] (2) Dissolve 0.4 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. While continuously stirring, add 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC) and 0.2 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution and continue stirring. The stirring speed is 600 rpm and the stirring time is 25 min to obtain mixed solution 1;

[0062] (3) Dissolve 0.4 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution. Add 0.008 g of zinc-doped mesoporous bioactive glass (ZnMBG) to the aqueous carboxylated chitosan solution and stir magnetically. The stirring speed is 400 rpm and the stirring time is 30 min to obtain mixed solution 2;

[0063] (4) Mix and stir mixed solution 1 and mixed solution 2, then pour them into a mold. The stirring speed is 400 rpm and the stirring time is 1 h. Let it stand at -20 °C for 36 h for preliminary cross-linking, and freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a cryogel with a macroporous structure;

[0064] (5) Immerse the obtained cryogel in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking, and freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge with shape memory function, denoted as SA / CCS / ZnMBG-1.

[0065] Example 2

[0066] Compared with Example 1, except that 0.02 g of zinc-doped mesoporous bioactive glass (ZnMBG) was added to the aqueous solution of carboxylated chitosan and stirred magnetically, other processes and parameters were the same as those in Example 1, denoted as SA / CCS / ZnMBG-2.

[0067] Example 3

[0068] Compared with Example 1, except that 0.04 g of zinc-doped mesoporous bioactive glass (ZnMBG) was added to the aqueous solution of carboxylated chitosan and stirred magnetically, other processes and parameters were the same as those in Example 1, denoted as SA / CCS / ZnMBG-3.

[0069] Example 4

[0070] Compared with Example 1, except that 0.08 g of zinc-doped mesoporous bioactive glass (ZnMBG) was added to the aqueous solution of carboxylated chitosan and stirred magnetically, other processes and parameters were the same as those in Example 1, denoted as SA / CCS / ZnMBG-4.

[0071] Example 5

[0072] A preparation method of a bioactive glass composite antibacterial hemostatic cryogel sponge, comprising the following steps:

[0073] (1) Dissolve 2.1 g of cetyltrimethylammonium chloride in 99 mL of deionized water, slowly add 30 mL of ethyl acetate under continuous stirring and continue stirring for 30 min to form uniform microemulsion droplets, with a stirring speed of 400 rpm, then slowly add 21 mL of 2 mol / L ammonia water and continue stirring for 15 min, with a stirring speed of 400 rpm, to obtain a mixture. Finally, add 12.4998 g of tetraethoxysilane, 1.4572 g of triethoxyphosphate, 7.3207 g of calcium nitrate tetrahydrate, and 1.4876 g of zinc nitrate hexahydrate to the obtained mixture at intervals of 30 min and continue stirring for 4 h, with a stirring speed of 400 rpm. Centrifuge to collect the product, with a centrifugation speed of 8000 revolutions per minute and a centrifugation time of 5 min. Wash the precipitate three times with alternating water and ethanol, dry the precipitate in an oven at 60 °C for 24 h, and then calcine it at 650 °C for 3 h to obtain zinc-doped mesoporous bioactive glass. The zinc-doped mesoporous bioactive glass is composed of the following components in molar percentages: SiO2 60%, P2O5 4%, CaO 31%, ZnO 5%, with a particle size of 100 - 400 nm and a mesoporous pore size of 2 - 15 nm;

[0074] (2) Dissolve 0.3 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. After adding 0.15 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.15 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution under continuous stirring, continue stirring at a stirring speed of 600 rpm for 25 min to obtain mixed solution 1;

[0075] (3) Dissolve 0.3 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution. Add 0.04 g of zinc-doped mesoporous bioactive glass (ZnMBG) to the aqueous carboxylated chitosan solution and stir magnetically at a stirring speed of 400 rpm for 30 min to obtain mixed solution 2;

[0076] (4) Mix and stir mixed solution 1 and mixed solution 2, then pour them into a mold. Stir at a stirring speed of 400 rpm for 1 h, and let it stand at -20 °C for 36 h for preliminary cross-linking. Freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a cryogel with a macroporous structure;

[0077] (5) Immerse the obtained cryogel in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking. Freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge with shape memory function, denoted as SA / CCS / ZnMBG-5.

[0078] Example 6

[0079] A preparation method of a bioactive glass composite antibacterial hemostatic cryogel sponge, comprising the following steps:

[0080] (1) Dissolve 2.1 g of cetyltrimethylammonium chloride in 99 mL of deionized water. Under continuous stirring, slowly add 30 mL of ethyl acetate and continue stirring for 30 min to form uniform microemulsion droplets. The stirring speed is 400 rpm. Then, slowly add 21 mL of 2 mol / L ammonia water and continue stirring for 15 min. The stirring speed is 400 rpm to obtain a mixture. Finally, add 12.4998 g of tetraethoxysilane, 1.4572 g of triethoxyphosphate, 7.3207 g of calcium nitrate tetrahydrate, and 1.4876 g of zinc nitrate hexahydrate to the obtained mixture at intervals of 30 min and continue stirring for 4 h. The stirring speed is 400 rpm. Centrifuge to collect the product. The centrifugation speed is 8000 revolutions per minute and the centrifugation time is 5 min. Wash the precipitate three times with alternating water and ethanol. Dry the precipitate in an oven at 60 °C for 24 h, and then calcine it at 650 °C for 3 h to obtain zinc-doped mesoporous bioactive glass. The zinc-doped mesoporous bioactive glass is composed of the following components in molar percentages: SiO₂ 60%, P₂O₅ 4%, CaO 31%, ZnO 5%. The particle size is 100 - 400 nm, and the mesoporous pore size is 2 - 15 nm;

[0081] (2) Dissolve 0.2 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. Under continuous stirring, add 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC) and 0.1 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution and continue stirring. The stirring speed is 600 rpm and the stirring time is 25 min to obtain mixed solution 1;

[0082] (3) Dissolve 0.2 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution. Add 0.04 g of zinc-doped mesoporous bioactive glass (ZnMBG) to the aqueous carboxylated chitosan solution and stir magnetically. The stirring speed is 400 rpm and the stirring time is 30 min to obtain mixed solution 2;

[0083] (4) Mix and stir mixed solution 1 and mixed solution 2 and then pour them into a mold. The stirring speed is 400 rpm and the stirring time is 1 h. Let it stand at -20 °C for 36 h for preliminary cross-linking, and freeze-dry it at -20 °C for 48 h. The vacuum degree is 10 Pa to obtain a cryogel with a macroporous structure;

[0084] (5) Immerse the obtained cryogel in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking, and freeze-dry it at -20 °C for 48 h. The vacuum degree is 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge with shape memory function, denoted as SA / CCS / ZnMBG-6.

[0085] Comparative Example 1

[0086] (1) Dissolve 0.4 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. After continuously stirring, add 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC) and 0.2 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution and continue stirring. The stirring speed is 600 rpm and the stirring time is 25 min to obtain Mixed Solution 1;

[0087] (2) Dissolve 0.4 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution;

[0088] (3) Mix and stir Mixed Solution 1 and the aqueous carboxylated chitosan solution, then pour them into a mold. The stirring speed is 400 rpm and the stirring time is 1 h. Let it stand at -20 °C for 36 h for preliminary cross-linking, and freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a cryogel;

[0089] (4) Immerse the obtained cryogel in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking, and freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge, denoted as SA / CCS-1.

[0090] Comparative Example 2

[0091] (1) Dissolve 0.3 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. After continuously stirring, add 0.15 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC) and 0.15 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution and continue stirring. The stirring speed is 600 rpm and the stirring time is 25 min to obtain Mixed Solution 1;

[0092] (2) Dissolve 0.3 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution;

[0093] (3) Mix and stir Mixed Solution 1 and the aqueous carboxylated chitosan solution, then pour them into a mold. The stirring speed is 400 rpm and the stirring time is 1 h. Let it stand at -20 °C for 36 h for preliminary cross-linking, and freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a cryogel;

[0094] (4) The obtained cryogel was immersed in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking, and freeze-dried at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge, denoted as SA / CCS-2.

[0095] Comparative Example 3

[0096] (1) Dissolve 0.2 g of sodium alginate (SA) in 10 mL of deionized water to obtain an aqueous sodium alginate solution. Under continuous stirring, add 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide chloride (EDC) and 0.1 g of N-hydroxysuccinimide (NHS) to the aqueous sodium alginate solution and continue stirring. The stirring speed is 600 rpm and the stirring time is 25 min to obtain a mixed solution 1;

[0097] (2) Dissolve 0.2 g of carboxylated chitosan (CCS) in 10 mL of deionized water to obtain an aqueous carboxylated chitosan solution;

[0098] (3) Mix and stir the mixed solution 1 and the aqueous carboxylated chitosan solution, then pour them into a mold. The stirring speed is 400 rpm and the stirring time is 1 h. Let it stand at -20 °C for 36 h for preliminary cross-linking, and then freeze-dry at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a cryogel;

[0099] (4) The obtained cryogel was immersed in an anhydrous calcium chloride solution with a mass concentration of 0.05 g / mL for 0.5 h for secondary cross-linking, and freeze-dried at -20 °C for 48 h with a vacuum degree of 10 Pa to obtain a bioactive glass composite antibacterial hemostatic cryogel sponge, denoted as SA / CCS-3.

[0100] Effect experiment

[0101] 1. Microscopic morphology of the cryogel sponge

[0102] The microscopic morphology of the zinc-doped bioactive glass (ZnMBG) in Example 1 was observed using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The results are shown in Figure 1 Figures a and b. The zinc-doped bioactive glass in Example 1 is well-dispersed nanoparticles, and there are a large number of nanopores on the surface, with a typical mesoporous structure. The microscopic morphology of the sodium alginate / carboxylated chitosan sponge in Comparative Example 1 and the sodium alginate / carboxylated chitosan / zinc-doped bioactive glass composite sponge in Example 1 was observed using a scanning electron microscope (SEM). The results are shown in Figure 2 Figures a and b. The sodium alginate / carboxylated chitosan sponge (SA / CCS-1) has a smooth surface and shows a highly interpenetrating macroporous structure; The results are shown inFigure 3 As shown in Figures a and b, zinc-doped bioactive glass is dispersed on the surface of sodium alginate / carboxylated chitosan / zinc-doped bioactive glass cryogel sponge (SA / CCS / ZnMBG-1), and it also exhibits an interpenetrating macroporous structure.

[0103] 2. Mechanical property testing of cryogel sponge

[0104] The cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3 were made into cylindrical samples with a diameter of 8 mm and a height of 5 mm, and their mechanical properties were tested using a dynamic thermomechanical analyzer. The compression strain was set to 80%, and the compression speed was 2 mm / min. Figure 4 Figures a and b are compression stress-strain curves.

[0105] Comparing the compression performance of Examples 1-6 and Comparative Examples 1-3, it can be seen that Examples 1-6 all have better mechanical properties and higher compression strength, and can generate sufficient mechanical pressure locally at the wound surface to resist blood flow impact and achieve physical hemostasis. The introduction of ZnMBG particles can be dispersed in the cryogel matrix and play a role in physical enhancement, significantly improving the mechanical properties of the cryogel.

[0106] Among them, after undergoing 10 compression cycles, Examples 3 and Comparative Example 1 can still maintain a stable structure and can return to the shape before compression after absorbing water, showing good shape memory. Figure 4 Figure c is the cyclic compression stress-strain curve.

[0107] Compared with Comparative Example 1, Example 3 shows better compression resistance and fatigue resistance. It can dissipate more mechanical energy along the macroporous structure channels during deformation, and at the same time can maintain its elastic properties and achieve effective shape recovery after compression. The introduction of ZnMBG enhances the energy dissipation of the composite cryogel, enabling it to better cope with incompressible wound bleeding.

[0108] 3. In vitro hemolysis experiment of cryogel sponge

[0109] Method for measuring in vitro hemolysis rate: To collect red blood cells, 5 ml of sheep blood containing sodium citrate anticoagulant was mixed with 5 ml of PBS, and then centrifuged at a centrifugal force of 200 g for 15 min. The obtained red blood cell suspension was diluted 10 times with PBS. Different concentrations of cryogel sponge suspensions with concentration values of 500, 1000, and 2000 μg / mL were prepared by placing the cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3 into PBS. The diluted RBCs were added to the cryogel sponge suspension. The positive control and negative control were PBS and deionized water, respectively. After incubation at 37 °C for 60 min, each sample was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0110] Figure 5 As a result of the in vitro hemolysis rate of the cryogel sponge, the hemolysis rate of all cryogel groups was less than 5%, that is, all were lower than the national medical biomaterial standard (<5%), indicating good blood compatibility.

[0111] 4. In vitro blood coagulation performance test of cryogel sponge

[0112] This experiment aimed to evaluate the ability of cryogel sponge to improve blood coagulation efficiency in vitro, and the index used was the whole blood coagulation index (BCI). First, cylindrical samples with a diameter of 9 mm and a height of 5 mm were prepared using commercial gauze, gelatin sponge, and the cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3. These samples were placed in a vial and preheated to 37 °C. Subsequently, 200 μl of recalcified whole blood (0.2 M calcium chloride: whole blood = 1:9) was added dropwise to the samples. The samples were incubated on a shaker at 37 °C and 50 rpm / min for 5 min. Then, 10 ml of deionized water was added to the samples to rupture the red blood cells in the unstable clots. Next, 100 μl of the upper layer solution was transferred to a 96-well plate. Using an enzyme-linked immunosorbent assay (ELISA) reader, the absorbance of hemoglobin at 540 nm was measured. The BCI index can be calculated by the following formula: BCI (%) = (I s -I0) / (I0-I r ). The lower the BCI value, the better the in vitro coagulation ability. The absorbance of the samples, the control group without added material, and the plate were represented by I s , I r , and I0, respectively.

[0113] The results are as Figure 6As shown, the cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3 can promote blood coagulation in vitro. Compared with Comparative Examples 1-3, the in vitro coagulation index of Examples 1-6 is lower and the in vitro coagulation ability is stronger. The introduction of ZnMBG can enhance the ability of the cryogel sponge to activate the intrinsic and extrinsic coagulation cascades, thereby enhancing the hemostatic effect of the material.

[0114] 5. Erythrocyte and platelet adhesion experiments of cryogel sponges.

[0115] Place 100 μL of the erythrocyte suspension on the surface of the SA / CCS / ZnMBG group cryogel sponge prepared in Example 3 and incubate at 37 °C for 5 min. Rinse the sample three times with PBS to remove loosely bound erythrocytes, and fix with 2.5% glutaraldehyde at 4 °C for 2 h. The sample was then dehydrated with a series of ethanol solutions and then observed by scanning electron microscopy (SEM). Place 20 μL of PRP on the surface of the SA / CCS / ZnMBG cryogel sponge and incubate at 37 °C for 5 min. Subsequently, rinse the sample three times with PBS to wash away physically adhered platelets, and fix with 2.5% glutaraldehyde at 4 °C for 2 h. After that, the sample was dehydrated with a series of ethanol solutions and then observed by scanning electron microscopy (SEM).

[0116] The results are as Figure 7 shown in Figures a and b below. The SA / CCS / ZnMBG-3 composite cryogel can adhere a large number of erythrocytes and platelets, and at the same time form a fibrin network, which can promote blood coagulation.

[0117] 6. Study the cytocompatibility of cryogel sponge extracts with L929 cells

[0118] Soak the cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3 with complete medium, and filter to obtain an extract of 20 mg / ml; culture L929 cells in complete culture medium containing 10% fetal bovine serum and antibiotics (penicillin 100 IU / mL, streptomycin 100 μg / mL), and place them in an incubator (conditions: 5% CO2, 37 °C, humidity > 90%). Digest the cells with 0.25% trypsin to prepare a cell suspension. Adjust the cell density to 1×10 5 cells / mL cell suspension. Inoculate the cell suspension into a 96-well culture plate, 100 μL per well, and culture in an incubator for 24 h. After the cells form a monolayer, discard the original culture medium, add 100 μL of the extract respectively, and continue to culture for 24 h. Take out the 96-well plate for cell morphology observation, remove the extract, add CCK-8 working solution, and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader. Among them, the control group is complete medium.

[0119] Figure 8 The results of cell compatibility show that different groups of cryogel sponges have good cell compatibility with L929 fibroblasts. Compared with comparative examples 1-3, examples 1-6 have higher absorbance, which means more cells, better cell compatibility, and the effect of promoting cell proliferation. The reason is that the introduction of ZnMBG releases bioactive ions such as silicon ions, calcium ions, and zinc ions, giving the material higher bioactivity.

[0120] 7. Antibacterial properties of cryogel sponges against different bacteria

[0121] This experiment aims to verify the antibacterial properties of the composite cryogel, and E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) are used for confirmation. The cryogel sponges prepared in Examples 1-6 and Comparative Examples 1-3 were cut into cylindrical shapes with a diameter of 9 mm and a height of 1 mm. 50 μl of 10 6 CFU·mL -1 To ensure that the material interacts fully with the bacteria, the sample was incubated at 37°C for 4 h. Subsequently, 950 μl of PBS solution was added to the sample and incubated for another 3 h to promote Zn 2+ Release. After shaking, 10 μl of the suspension was evenly spread on an agar plate and incubated at 37°C for 12 h, and then the number of bacterial colonies was counted. Bactericidal rate (%) = (number of bacteria in the blank group - number of bacteria in the experimental group) / number of bacteria in the blank group * 100.

[0122] like Figure 9 As shown in Figure 2, Examples 1-6 showed better antibacterial effects than Comparative Examples 1-3, and the antibacterial effects were significantly enhanced with the introduction of zinc ions. The antibacterial properties of the composite cryogels were mainly attributed to the presence of amino groups in CCS and the presence of Zn 2+ The release of positively charged amino groups and Zn 2+ It can attract negatively charged bacteria, destroy the integrity of bacterial cell membranes and change their permeability, leading to bacterial death.

[0123] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge, characterized in that: The following steps are involved: (1) dissolving sodium alginate in deionized water to obtain a sodium alginate aqueous solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carboxyl chloride and N-hydroxysuccinimide to the sodium alginate aqueous solution under continuous stirring, and continuing to stir to obtain a mixed solution 1; (2) dissolving carboxylated chitosan in deionized water to obtain a carboxylated chitosan aqueous solution, adding zinc-doped mesoporous bioactive glass to the carboxylated chitosan aqueous solution and dispersing the glass uniformly to obtain a mixed solution 2; (3) Mixing and stirring the mixed solution 1 and the mixed solution 2, pouring the mixture into a mold, standing at low temperature for preliminary cross-linking, and freeze-drying to obtain a frozen gel; (4) Soaking the obtained cryogel in an anhydrous calcium chloride solution for secondary crosslinking, and freeze-drying to obtain the bioactive glass composite antibacterial hemostatic cryogel sponge.

2. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (1), the mass to volume ratio of sodium alginate, 1-ethyl-3-(3-dimethylaminopropyl)carboxyl chloride, N-hydroxysuccinimide and deionized water is (0.1-0.5) g:(0.05-0.25) g:(0.05-0.25) g:10 mL.

3. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (2), the mass volume ratio of carboxylated chitosan, zinc-doped mesoporous bioactive glass and deionized water is (0.1-0.5) g: (0.008-0.1) g: 10 mL.

4. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (2), the zinc-doped mesoporous bioactive glass is composed of the following components in molar percentage: SiO2 60%, P2O 54%, CaO 31%, ZnO 5%, with a particle size of 100-400nm and a mesopore diameter of 2-15nm.

5. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (3), the method for preparing the zinc-doped mesoporous bioactive glass comprises the following steps: Dissolve hexadecyltrimethylammonium chloride in deionized water, slowly add ethyl acetate under continuous stirring and continue stirring, then slowly add ammonia water and continue stirring to obtain a mixture, finally add tetraethoxysilane, triethoxyphosphoric acid, calcium nitrate tetrahydrate and zinc nitrate hexahydrate to the obtained mixture in sequence after a period of time and continue stirring, collect the product by centrifugation, wash the precipitate with alternating water and ethanol for multiple times, dry the precipitate, and calcine to obtain zinc-doped mesoporous bioactive glass.

6. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (3), the mass ratio of sodium alginate in the mixed solution 1 to carboxylated chitosan in the mixed solution 2 is 1:

1.

7. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (3), the mixture is allowed to stand at a temperature of -20 to -80°C for 24 to 48 hours, the freeze-drying temperature is -20 to -80°C, the freeze-drying time is 24 to 48 hours, and the vacuum degree is 1 to 15 Pa.

8. The method for preparing a bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 1, characterized in that: In step (4), the mass concentration of the calcium chloride solution is 0.01-0.05 g / mL, the soaking time is 0.5-1.5 h, the freeze-drying temperature is -20--80° C., the freeze-drying time is 24-48 h, and the vacuum degree is 1-15 Pa.

9. A bioactive glass composite antibacterial hemostatic cryogel sponge prepared by the method according to any one of claims 1 to 8.

10. Use of the bioactive glass composite antibacterial hemostatic cryogel sponge according to claim 9 in the preparation of incompressible wound hemostasis and infection control medical materials.

Images