Expansion type efficient hemostatic sponge and preparation method thereof

Abstract

The invention relates to an intumescent efficient hemostatic sponge and a preparation method thereof, the preparation method comprises the following steps: preparation of HAMA: utilizing hyaluronic acid to react with methacrylic anhydride, and grafting double bonds on a hyaluronic acid macromolecular skeleton; preparing HAMA hydrogel: pouring the HAMA solution dissolved with the photoinitiator into a mold, and initiating crosslinking in an ultraviolet radiation manner to obtain the HAMA hydrogel; the HAMA hydrogel is taken out of the mold and dried, and the hemostatic sponge is formed. The expansion type efficient hemostatic sponge can be prepared by adopting the preparation method. The hemostatic sponge disclosed by the invention shows remarkable advantages in the aspects of expansion ratio, hemostatic effect, biocompatibility and the like, and has a wide application prospect and an important clinical value. The preparation method disclosed by the invention is easy to operate in process flow and suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical hemostatic materials, and particularly relates to an expanded high-efficiency hemostatic sponge and a preparation method thereof. BACKGROUND

[0002] As a key consumable in surgical operations, trauma first aid and battlefield rescue, the performance of medical hemostatic materials directly affects the prognosis and life safety of patients. Hemostatic sponge, as a widely used hemostatic material, usually achieves hemostasis function through physical compression, liquid absorption expansion or chemical coagulation mechanism. Traditional hemostatic sponge is usually based on natural high molecular materials (such as gelatin, collagen) or synthetic polymers as matrix, and constructs porous structure through cross-linking solidification, freeze-drying and other processes to enhance its liquid absorption capacity and mechanical strength. In the preparation process, the hydrophilicity, porosity and cross-linking density of the material are the core parameters affecting its hemostatic efficiency, and the drying method (such as freeze-drying, critical point drying) further regulates the stability of its three-dimensional structure and the expansion property.

[0003] CN114470305A discloses a hemostatic sponge and a preparation method thereof. The components for preparing the hemostatic sponge comprise, by mass: 3-30 parts of a compound containing double bonds, 1-10 parts of chitosan, and 0.1-15 parts of a photoinitiator. The hemostatic sponge of the present application has higher water absorption rate and water absorption rate than existing products, and can rapidly expand after absorbing water. When applied to wound hemostasis, it can quickly seal the wound and stop bleeding.

[0004] CN110522945A discloses a medical biological gel hemostatic dressing and a preparation method thereof. The present application first uses cocoon and polyvinyl alcohol solution to prepare a silk fibroin mixed solution, then mixes bacterial cellulose with the silk fibroin mixed solution and adds polyacrylamide, and after freeze-thaw cycle, a gel blank is prepared; then the gel blank is treated with a chitosan quaternary ammonium salt treatment solution, and then mixed with a silver ammonia solution containing sodium alginate and a calcium ion solution, filtered, freeze-dried to obtain a medical biological gel hemostatic dressing.

[0005] Although the existing hemostatic sponge meets the clinical needs to some extent, it still has significant limitations in actual application. For example, the traditional hemostatic material has a slow water absorption and expansion rate, and it is difficult to quickly form an effective compression barrier in complex wounds (such as internal bleeding or multiple trauma), which limits the hemostatic efficiency. In addition, the liquid absorption rate and volume expansion rate of the existing hemostatic sponge are often insufficient, and it is easy to fall off due to mechanical external force or body fluid flow, increasing the risk of secondary bleeding. At the same time, the degradation rate of some materials in the body does not match the tissue repair period, which may cause chronic inflammation or foreign body reaction. The above problems limit the universality and reliability of the hemostatic sponge in emergency scenes, and it is urgent to develop a new type of hemostatic material with high liquid absorption and expansion performance, excellent biocompatibility and controllable degradation ability.

[0006] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, a large number of literatures and patents are studied by the applicant when the invention is made, but due to the limitation of space, all the details and contents are not listed in detail, but this does not mean that the invention does not have these prior art characteristics, on the contrary, the invention has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides an expanded high-efficiency hemostatic sponge and a preparation method thereof to solve at least part of the above technical problems. Compared with the prior art, the hemostatic sponge prepared by the preparation method of the present application has significant advantages in expansion ratio, hemostatic effect, biocompatibility and the like, and has wide application prospect and important clinical value. In addition, the process flow of the preparation method of the present application is easy to operate and suitable for large-scale production.

[0008] The present application discloses a preparation method of an expanded high-efficiency hemostatic sponge, which comprises the following steps:

[0009] Preparation of HAMA: reacting hyaluronic acid with methacrylic anhydride to graft double bonds on the hyaluronic acid macromolecular backbone;

[0010] Preparation of HAMA hydrogel: pouring the HAMA solution dissolved with a photoinitiator into a mold, and initiating crosslinking by ultraviolet irradiation to obtain HAMA hydrogel;

[0011] The HAMA hydrogel is taken out of the mold and dried to form a hemostatic sponge.

[0012] The present application solves the technical problems of poor hydrophilicity, low liquid absorption rate and insufficient expansion ratio of traditional hemostatic sponge by reacting hyaluronic acid (HA) with methacrylic anhydride (MA) to graft double bonds on the HA macromolecular backbone to generate methacrylated hyaluronic acid (HAMA). The double bond structure of HAMA endows it with excellent photo-crosslinking ability, and combined with the ultraviolet light-induced crosslinking process, a highly crosslinked three-dimensional network structure can be formed. This network structure retains the porous morphology through physical compression or freeze-drying during the subsequent drying process, thereby significantly improving the liquid absorption rate and volume expansion rate of the hemostatic sponge. Experiments show that the volume expansion rate of the HAMA-based hemostatic sponge can break through 1200% within 30 minutes, which is much higher than the 300% of the commercially available product. This feature enables it to quickly form a physical compression barrier in complex wounds, while removing blood from the wound surface through high liquid absorption capacity, reducing the risk of infection and promoting the initiation of the coagulation cascade, ultimately achieving efficient hemostasis.

[0013] According to a preferred embodiment, in the preparation step of HAMA, hyaluronic acid is dissolved in a mixed solvent composed of an organic solvent and water, stirred until completely dissolved; the solution is placed in an ice bath environment, and methyl acrylate is slowly added dropwise, and the reaction is continued under ice bath conditions; after the reaction is completed, the pH of the system is adjusted by alkaline conditions, and the product is precipitated by adding a precipitant, and the HAMA crude product is obtained by centrifugal separation.

[0014] In the preparation step of HAMA, the ice bath environment is used to slowly add MA and adjust the pH to alkaline, which effectively solves the problem of HA degradation or residual by-products in the traditional chemical modification process. The low-temperature reaction condition (ice bath) can inhibit the HA chain breakage caused by excessive initiation of free radicals, ensuring the stability of the molecular weight of HAMA; after adjusting the pH of the system under alkaline conditions, anhydrous ethanol is added as a precipitant, and the unreacted MA and by-products can be efficiently removed by centrifugal separation to purify the HAMA crude product. This process optimizes the chemical uniformity of HAMA, avoids the interference of impurities in the subsequent cross-linking reaction, and thus ensures the uniformity and mechanical strength of the hydrogel network structure. The three-dimensional network formed after the purified HAMA is cross-linked under ultraviolet light is more compact, further improving the liquid absorption rate and swelling efficiency of the hemostatic sponge, and laying a foundation for the stability of the subsequent hemostatic performance.

[0015] According to a preferred embodiment, the prepared HAMA crude product is dissolved and loaded into a dialysis bag, and the unreacted methyl acrylate and by-products are removed by dialysis, and then subjected to sterile filtration treatment and dried to obtain a HAMA solid sample.

[0016] Loading the HAMA crude product into a dialysis bag and removing the unreacted MA and by-products by dialysis solves the problem of decreased biocompatibility of traditional hemostatic materials caused by residual chemical reagents. The dialysis process uses a dialysis bag with a molecular weight cutoff of 50 kDa, which can effectively remove small molecular impurities while retaining the macromolecular backbone of HAMA. Subsequent sterile filtration treatment (0.22 μm filter membrane) further removes microbial contamination, ensuring the sterility of the hemostatic sponge. The HAMA solid sample obtained by freeze-drying has a regular porous structure, providing a stable physical basis for the subsequent cross-linking and drying of the hydrogel. This purification and drying process significantly reduces the cytotoxicity risk of the hemostatic sponge, and by retaining the natural biocompatibility of HA, it reduces the inflammatory response when contacting the wound, improving the safety of clinical application.

[0017] According to a preferred embodiment, the mixed solvent is a mixed system of dimethylformamide and water; the precipitant is anhydrous ethanol, which is compatible with HAMA to efficiently separate the target product; after sterile filtration treatment, a freeze-drying process is used to optimize the morphology and pore structure of HAMA.

[0018] The mixed solvent of dimethylformamide (DMF) and water is used to dissolve HA, which solves the problem of poor solubility or insufficient reactivity in traditional organic solvent system. DMF as a polar solvent can effectively disperse HA molecules, while the presence of water maintains the hydrophilicity of the system and promotes the uniform grafting reaction of MA and HA. Anhydrous ethanol as a precipitant has good compatibility with HAMA, which can efficiently separate the target product and avoid the structural damage of HAMA that may be caused by traditional organic solvents such as acetone. The freeze-drying process retains the three-dimensional porous structure of HAMA by pre-freezing and low-pressure drying, avoiding the network collapse caused by high temperature. This combined process not only optimizes the morphology and porosity of HAMA, but also ensures the high permeability and volume expansion capacity of the subsequent hydrogel during liquid absorption, thereby improving the clinical applicability of hemostatic sponges.

[0019] According to a preferred embodiment, the HAMA solid is re-dissolved in deionized water or phosphate buffer, a biocompatible photoinitiator is added, and after stirring to complete dissolution, the mixture is injected into a mold; a UV light source is used to initiate crosslinking, and after crosslinking is completed, the hydrogel is taken out of the mold and left to promote the stabilization of the crosslinked network.

[0020] The HAMA solid is dissolved in deionized water or PBS, and a biocompatible photoinitiator (such as LAP, TPO-L) is added, which solves the problem of toxicity or uneven crosslinking of the photoinitiator in traditional UV crosslinking systems. The selection of photoinitiator takes into account biocompatibility and crosslinking efficiency, ensuring rapid crosslinking of HAMA double bonds under UV irradiation to form a stable network. The pretreatment of the mold (silicone oil or polytetrafluoroethylene coating) reduces the demolding resistance and avoids the destruction of the hydrogel structure during removal. The standing treatment after crosslinking (37℃ incubator) can promote the stress release and stabilization of the crosslinked network, improving the mechanical strength of the hydrogel. This process optimizes the uniformity and physical integrity of the hydrogel, providing a key guarantee for the preservation of the porous structure and the exertion of hemostatic performance in the subsequent drying step.

[0021] According to a preferred embodiment, the concentration of HAMA in the mixture is 5% to 10%, more preferably 10%.

[0022] The concentration of the HAMA solution is controlled at 5% to 10%, which solves the problem of imbalance between liquid absorption rate and mechanical strength caused by too low or too high crosslinking network density of traditional hemostatic sponges. The moderate three-dimensional network density formed after ultraviolet crosslinking of 10% HAMA concentration provides sufficient hydrophilic channels to accommodate liquid and supports efficient volume expansion through moderate crosslinking. Experiments show that the liquid absorption rate of the 10% HAMA group can reach more than 4000% within 20 minutes, while the liquid absorption capacity of the 5% HAMA group is significantly reduced due to the sparse crosslinking network. The concentration gradient control is directly related to the physical properties and hemostatic efficiency of the hemostatic sponge, enabling it to quickly absorb blood from the wound and apply continuous pressure to the wound through volume expansion, thereby achieving the synergistic effect of active hemostasis and physical compression.

[0023] According to a preferred embodiment, the biocompatible photoinitiator comprises lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, 2,4,6-trimethylbenzoyl phenyl ethyl phosphonate or Irgacure 2959.

[0024] Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 2,4,6-trimethylbenzoyl phenyl ethyl phosphonate (TPO-L) or Irgacure 2959 are selected as photoinitiators, which solves the problem of unstable performance of traditional hemostatic sponges caused by toxicity or low crosslinking efficiency of photoinitiators. The above initiators have high ultraviolet absorption capacity and low cytotoxicity, can quickly initiate crosslinking of HAMA double bonds in the wavelength range of 254-365 nm, and form a uniform three-dimensional network. Compared with non-biocompatible initiators (such as azo compounds), the selected initiators have almost no residual toxicity after crosslinking, avoiding chemical irritation to the wound tissue. In addition, they have high initiation efficiency, can shorten the ultraviolet irradiation time, reduce the risk of HA degradation, and thus ensure the biocompatibility and hemostatic efficiency of the hemostatic sponge.

[0025] According to a preferred embodiment, drying is carried out in two steps: first, the hydrogel is dried in a constant temperature oven, and then the dried hydrogel is freeze-dried in a freeze-dryer to preserve the internal pore structure.

[0026] The two-step drying method (preliminary drying and then freeze drying) solves the problem of uneven pore distribution or insufficient mechanical strength in traditional single drying process (such as freeze drying). The preliminary drying (50-80 DEG C, 40 min) can quickly remove the surface moisture to form a preliminary porous network, and the subsequent freeze drying (-40 DEG C to -80 DEG C) can retain the internal pore structure and avoid collapse. The two-step method combines the advantages of rapid dehydration of drying and the structural stability of freeze drying, so that the hemostatic sponge can realize efficient expansion through the elastic expansion of the cross-linked network when absorbing liquid. Experiments show that the liquid absorption rate of the hemostatic sponge prepared by this method is more than 30% higher than that of the single drying process, and the mechanical strength is significantly enhanced, thereby reducing the risk of material shedding caused by external force in complex trauma scenarios and improving the reliability of hemostasis.

[0027] According to a preferred embodiment, after the cross-linked hydrogel is taken out of the mold, it is subjected to mechanical compression treatment before drying: the hydrogel is placed in a hydraulic device or a mechanical press, and a preset pressure is continuously applied, so that the pore structure is adjusted by physical compression, and the cross-linked network forms a denser elastic matrix to improve the volume expansion multiple when absorbing water.

[0028] The mechanical compression treatment of the hydrogel before drying solves the problem of insufficient liquid absorption expansion multiple caused by loose pore structure of the traditional hemostatic sponge. By applying a preset pressure through a hydraulic device or a press, the initial pore density of the hydrogel can be adjusted, so that the cross-linked network forms a denser elastic matrix. After compression treatment, the cross-linked network has significantly enhanced elastic expansion capacity when absorbing liquid, thereby realizing higher volume expansion multiple. This process optimizes the physical properties of the hemostatic sponge, enabling it to exert stronger mechanical compression on the wound in a short time, accelerating the hemostasis process and reducing the risk of secondary bleeding.

[0029] The application also discloses an expanded high-efficiency hemostatic sponge prepared by the preparation method.

[0030] The expanded high-efficiency hemostatic sponge prepared by the application combines the advantages of chemical modification of HAMA, ultraviolet cross-linking and two-step drying process, and solves the multiple technical problems of slow liquid absorption rate, low expansion multiple and poor biocompatibility of existing hemostatic materials. The core lies in that through the double bond modification and precise concentration regulation of HAMA, a cross-linked network with high hydrophilicity and high mechanical strength is constructed; and then combined with freeze drying and mechanical compression treatment, the porous structure is retained and the elastic expansion capacity is enhanced. The final product shows significantly better hemostatic efficiency (hemostatic time < 100 seconds) than the commercial product in the liver damage model, and its excellent biocompatibility is verified by Calcein-AM / PI staining (the proportion of living cells is > 95%). This technical scheme provides a new medical material solution for trauma first aid, which has the advantages of rapid hemostasis, biological safety and controllable degradation.

[0031] The component of the hemostatic sponge disclosed in the prior art (such as CN114470305A) comprises 3-30 parts of a double bond compound, 1-10 parts of chitosan, and 0.1-15 parts of a photoinitiator, and a double network structure is formed through vacuum reduction and light-induced crosslinking. The technical scheme of the present application is significantly different from that of the prior art. The present application discards the addition of chitosan and only uses methacrylated hyaluronic acid (HAMA) as the core component, and directly constructs a single chemical network structure through ultraviolet light-induced crosslinking. The difference in components and processes brings significant beneficial technical effects. The experimental data of the present application show that when chitosan is introduced into the HAMA system (10% HAMA+4% CS group), the intermolecular interaction (such as hydrogen bond, electrostatic adsorption) between chitosan and HAMA will limit the swelling freedom of HAMA chains, resulting in a significant decrease in liquid absorption rate and volume expansion rate; while the pure 10% HAMA system through optimized crosslinking network density and two-step drying process, its liquid absorption rate can reach more than 4000% within 20 minutes, and the volume expansion rate breaks through 1200% within 30 minutes, which is significantly better than the system containing chitosan and the commercial product. In terms of hemostatic effect, the pure HAMA sponge of the present application removes blood from the wound surface through rapid liquid absorption (reduces the risk of infection) and volume expansion to form sustained physical compression (promotes blood vessel closure), which shows shorter hemostatic time in the mouse liver damage model. The technical effect benefits from the synergy of the optimization of pure HAMA component, concentration regulation and process design. Compared with the technical path of the prior art which relies on the coagulation effect of chitosan, the present application has obvious advantages in liquid absorption and expansion performance and hemostatic efficiency. In summary, through the design of the pure HAMA system, the present application solves the technical bottleneck of poor material compatibility and performance compromise in the traditional double network structure, realizes the synergistic optimization of water absorption rate, expansion rate and hemostatic efficiency, and provides a more reliable hemostatic solution for clinical emergency scenes. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a preparation method flow chart of the expanded high-efficiency hemostatic sponge provided by the present application.

[0033] Figure 2 is a liquid absorption rate comparison chart of a plurality of hemostatic sponges in an embodiment provided by the present application.

[0034] Figure 3 is a volume expansion rate comparison chart of a plurality of hemostatic sponges in an embodiment provided by the present application.

[0035] Figure 4 is a comparison chart of mouse liver bleeding hemostatic time of a plurality of hemostatic sponges in an embodiment provided by the present application.

[0036] Figure 5 is a biocompatibility characterization comparison chart of a plurality of hemostatic sponges in an embodiment provided by the present application.

[0037] Figure 6 is a contrast chart of in vivo degradability characterization of various hemostatic sponges in one embodiment provided by the present application. DETAILED DESCRIPTION

[0038] The application will be described in detail below with reference to the accompanying drawings.

[0039] The present application uses hyaluronic acid (HA) as raw material, by double bond treatment of hyaluronic acid, to obtain methacrylated hyaluronic acid (HAMA), and then to obtain sodium hyaluronate hydrogel by using ultraviolet light to initiate crosslinking, and to obtain an expanded high-efficiency hemostatic sponge by a two-step drying method.

[0040] Preferably, as shown in the figure, the preparation method of the expanded high-efficiency hemostatic sponge of the present application can include the following steps: Figure 1

[0041] S1, preparation of methacrylated hyaluronic acid (HAMA): using hyaluronic acid (HA) to react with methacrylic anhydride (MA) to graft double bonds on the HA macromolecular backbone;

[0042] S2, preparation of HAMA hydrogel: pour the HAMA solution containing a photoinitiator into a mold of a specific size and shape, and initiate crosslinking by ultraviolet irradiation to obtain HAMA hydrogel;

[0043] S3, take out the HAMA hydrogel from the mold and dry it to form a hemostatic sponge.

[0044] Preferably, in step S1, hyaluronic acid (HA) can be dissolved in a mixed solvent composed of an organic solvent and water, and the HA is completely dissolved under stirring conditions. Then, the solution is placed in a low-temperature environment, and methacrylic anhydride (MA) is slowly added dropwise, and the reaction is continued under low-temperature conditions to graft double bonds on the HA macromolecular backbone to generate HAMA. After the reaction is completed, the pH of the system is adjusted by alkaline conditions, a precipitant is added for product precipitation, and the HAMA crude product is obtained by centrifugal separation. The crude product is dissolved and loaded into a dialysis bag, and the unreacted MA and by-products are removed by dialysis, and after sterile filtration treatment, freeze-drying is performed to obtain a solid sample of HAMA.

[0045] Preferably, the mixing ratio of the organic solvent and water can be adjusted according to actual needs, for example, a mixed system of common organic solvents such as dimethylformamide (DMF) and water is used to balance the solubility and reactivity of HA.

[0046] Preferably, low-temperature reaction conditions (such as ice bath) can ensure that the reaction proceeds gently and avoid side reactions or HA degradation.

[0047] ​Preferably, the choice of precipitants should be compatible with HAMA, such as anhydrous ethanol or other organic solvents, to efficiently separate the target product.

[0048] Preferably, the steps of dialysis and filtration are aimed at purifying HAMA, removing residual reactants and impurities, and ensuring the stability of subsequent cross-linking and hemostatic performance.

[0049] Preferably, the freeze-drying process can optimize the morphology and pore structure of HAMA, providing a basis for subsequent preparation of hemostatic sponges.

[0050] Illustratively, in one preparation experiment of methacrylated hyaluronic acid (HAMA), 1 g of hyaluronic acid can be dissolved in a mixed solvent composed of 30 mL of dimethylformamide (DMF) and 60 mL of water, stirred at a speed of 400 rpm at room temperature until completely dissolved. Subsequently, the solution is placed in an ice bath environment, and 0.75-1.0 mmol (about 75-100 μL, MA density 1.04 g / mL) of methacrylic anhydride (MA) is slowly added dropwise, and the reaction is continued for 24 hours to graft double bonds onto the HA macromolecular backbone to generate HAMA. After the reaction is completed, the pH of the system is adjusted to 8-9 using sodium hydroxide, 3 times the volume of pre-cooled anhydrous ethanol is added for precipitation, and the precipitate is collected by centrifugation (5000 rpm, 10 minutes). The crude HAMA product is dissolved in phosphate buffered saline (PBS) and loaded into a dialysis bag with a molecular weight cut-off of 50 kDa, and dialyzed against deionized water for 3 days (3 times a day), to remove unreacted MA and by-products. The HAMA solution after dialysis is filtered through a 0.22 μm filter membrane and freeze-dried for 3 days to obtain a HAMA solid sample.

[0051] Preferably, in step S2, the HAMA solid can be re-dissolved and a biocompatible photoinitiator can be added, and after stirring uniformly, it can be injected into a mold of a specific shape, wherein the added initiator can include but is not limited to lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 2,4,6-trimethylbenzoyl phenyl ethyl phosphonate (TPO-L), or Irgacure 2959, etc.

[0052] Preferably, the HAMA solid can be dissolved in deionized water or phosphate buffered saline (PBS).

[0053] Preferably, after stirring until completely dissolved, the mixture can be injected into a mold treated with silicone oil or polytetrafluoroethylene coating to reduce adhesion during subsequent demolding.

[0054] Preferably, the UV cross-linking process can use a UV light source (such as a high-pressure mercury lamp or an LED UV lamp) with a wavelength of 254-365 nm, and the irradiation intensity is controlled within a predetermined range (such as 10-50 mW / cm 2), the irradiation time can be adjusted according to the type of photoinitiator and the concentration of HAMA. During the crosslinking process, it is necessary to ensure that the ultraviolet light uniformly penetrates the solution, and if necessary, the uniformity of crosslinking is optimized by rotating the mold or stepwise irradiation. After the crosslinking is completed, the hydrogel is taken out of the mold and placed in a suitable temperature environment for a certain period of time to promote the stabilization of the crosslinking network.

[0055] Preferably, in step S3, the crosslinked hydrogel is taken out of the mold and dried, wherein various drying methods can be used to dry the hydrogel, including but not limited to oven drying, freeze drying, critical point drying, etc.

[0056] Preferably, after the crosslinked hydrogel is taken out of the mold, the drying method can be selected according to the performance requirements of the target hemostatic sponge, wherein one of the above drying methods or a combination thereof can be selected to dry the hydrogel.

[0057] Preferably, the oven drying method is to place the hydrogel in a constant temperature oven, the temperature can be controlled within a predetermined range (such as 50-80°C), and the time can be adjusted according to the thickness of the hydrogel. This drying method can preliminarily remove part of the water to form a porous network structure, but attention should be paid to avoid excessive shrinkage of the crosslinking network or degradation of HA caused by too high temperature.

[0058] Preferably, freeze drying is to place the hydrogel in a freeze dryer, control the pre-freezing temperature and drying pressure within a predetermined range (such as -40℃ to -80℃ and 0.1 to 0.5 mbar), and continuously dry. This drying method can preserve the three-dimensional porous structure of the hydrogel and prevent collapse, but may cause uneven distribution of pores when used alone.

[0059] Preferably, critical point drying is to realize tension-free drying at critical temperature (31.1°C) and critical pressure (7.39 MPa) through carbon dioxide critical point drying technology (CO2 supercritical drying), which is suitable for scenarios that require high porosity and smoothness of the surface, but the equipment cost is high.

[0060] Further preferably, a two-step drying method of first oven drying for 40 min and then freeze drying can be used for processing to obtain a hemostatic sponge with high water absorption and expansion rate. This method combines the advantages of rapid dehydration of oven drying and the structural stability of freeze drying, which can significantly improve the water absorption and expansion rate of the hemostatic sponge and mechanical strength, while reducing the risk of pore collapse.

[0061] Preferably, to enhance the water absorption and swelling performance, the hydrogel can be subjected to mechanical compression treatment before drying: the hydrogel is placed in a hydraulic device or mechanical press plate, and a certain pressure is continuously applied. This treatment step can regulate the pore structure by physical compression, so that the cross-linked network forms a denser elastic matrix, thereby producing a higher volume expansion ratio when absorbing water. The compression parameters can be optimized according to the initial thickness of the hydrogel and the target expansion rate, for example, the thicker the hydrogel, the compression pressure can be appropriately reduced to avoid excessive densification.

[0062] Further, the water absorption performance of the hemostatic sponge is tested by specific gravity method: first, the hemostatic sponge is weighed, and the initial mass is recorded as M1; then the hemostatic sponge sample to be tested is soaked in simulated blood. After 2 min, 5 min, 10 min, 20 min and 30 min, it is taken out, weighed without excess water on the surface of the hemostatic sponge, and the result is recorded as M2. The liquid absorption rate of the hemostatic sponge is calculated using the following formula:

[0063] W = (M2-M1) / M1 x 100%,

[0064] Wherein, W represents the liquid absorption rate, M1 represents the mass of the freeze-dried hemostatic sponge, and M2 represents the mass of the hemostatic sponge after absorbing liquid.

[0065] Further, the volume expansion rate test is as follows: first, the diameter and height of the hemostatic sponge are measured with a vernier caliper, and the volume is calculated, denoted as V1. Then, after soaking in deionized water for 2 min, 5 min, 10 min, 20 min and 30 min, the hemostatic sponge is taken out and the diameter and height are measured again to calculate the volume again, denoted as V2. The swelling rate (L) is calculated by the following formula:

[0066] L = (V2-V1) / V1 x 100%,

[0067] Wherein, L is the swelling rate of the hemostatic sponge; V1 is the volume of the dried hemostatic sponge; V2 is the volume of the hemostatic sponge after absorbing water.

[0068] The expanded high-efficiency hemostatic sponge of the present application has a liquid absorption rate (such as shown in Figure 2 and a volume expansion rate (such as shown in Figure 3Test verification: 10% methacrylated hyaluronic acid (HAMA) group showed the best performance in liquid absorption and swelling capacity. The liquid absorption rate increased rapidly with time, reaching more than 4000% within 20 minutes and tending to be stable. The volume expansion rate also increased synchronously, breaking through 1200% within 30 minutes. In contrast, the 5% HAMA group had lower crosslinking network density, and its liquid absorption and swelling capacity were significantly weaker than that of the 10% HAMA group. In the 10% HAMA + 4% chitosan (CS) group, the intermolecular interactions (such as hydrogen bonds and electrostatic adsorption) between chitosan and HAMA limited the swelling freedom of HAMA chains, resulting in a significant decrease in liquid absorption rate and expansion rate, which confirmed the design advantage of using pure HAMA system in the present application. The liquid absorption rate of the commercial control product was less than 1000% for a long time, and the volume expansion rate was less than 300%, reflecting its limitations in porous structure construction or material hydrophilicity design.

[0069] From a technical mechanism perspective, the HAMA concentration directly regulates the crosslinking network density: after UV crosslinking, the three-dimensional network formed by 10% HAMA provides sufficient hydrophilic channels to accommodate liquid and supports efficient volume expansion through moderate network density. The preparation process (double bond modification, UV crosslinking, "baking + freeze-drying" two-step method) further guarantees the performance: baking quickly removes surface moisture, and freeze-drying preserves the internal pore structure, allowing the sponge to achieve efficient liquid permeation and volume expansion through network swelling when absorbing water.

[0070] The above performance enables the hemostatic sponge of the present application to remove blood from the wound surface by quickly absorbing liquid (reducing the risk of infection) and form sustained physical pressure on the wound surface relying on volume expansion (promoting blood vessel closure), which is significantly superior to existing commercial products in technical effect, fully embodying the synergistic effect of component optimization (pure HAMA), concentration regulation (10% HAMA), and process design (crosslinking and step drying).

[0071] Further, the hemostatic ability of the hemostatic sponge can be evaluated by the following experiment: the experimental animal is a c57 mouse with a body weight of about 30g, and a liver damage experiment is performed. The abdominal surface is cut open with tissue scissors at the xiphoid position, and the epithelial layer is clamped with tissue forceps, and the muscle and fascia are cut layer by layer. Open the abdominal cavity, wipe off the excess blood with gauze, and remove the liver from the abdominal cavity to fully expose it. Place clean filter paper (weighed in advance) and cut gauze under the liver. Use a scalpel to make a 3mm long and 2mm deep wound, and let it bleed freely for 2s. Then immediately insert the hemostatic sponge into the wound, and after 60s, gently remove the hemostatic sponge, and observe the wound condition. If there is continued bleeding, repeat the insertion of the sponge, and then observe every 15s, and repeat the above operation until there is no active bleeding from the wound. For the control group, let it bleed freely. Record the bleeding time and bleeding volume. For the positive control group, replace the hemostatic sponge with gauze for the experiment. Record the bleeding time and bleeding volume.

[0072] The present application evaluates the in vivo hemostatic efficiency of the hemostatic sponge through a mouse liver damage model, which simulates the clinical scenario of parenchymal organ trauma bleeding: a 3mm-long and 2mm-deep liver wound is constructed through a standardized operation, and a blank control group (free bleeding), a gauze group (a commonly used clinical dressing), a commercial hemostatic sponge control group and a 10% HAMA hemostatic sponge group are set up, and the time from active bleeding to complete hemostasis is recorded. From Figure 4 It can be seen that, compared with the blank control group (≈400 seconds), the gauze group (≈230 seconds) and the commercial control group (≈120 seconds), the 10% HAMA group has the shortest hemostatic time (mean <100 seconds), and there is a statistical difference among the four groups (**** represents P<0.0001, * represents P<0.05). Combined with the previous liquid absorption rate and volume expansion rate test results, its hemostatic mechanism can be analyzed as follows: the 10% HAMA sponge relies on high liquid absorption rate to quickly remove free blood from the wound (avoiding dilution of coagulation factors and accelerating the start of the coagulation cascade), while the rapid volume expansion forms a sustained mechanical compression on the wound (promoting the closure of the blood vessel stump); in addition, the biocompatibility of hyaluronic acid can reduce local inflammatory response and assist the stable adhesion of blood clots. Further verification by gross observation of the wound: after the 10% HAMA group is attached to the wound, blood is quickly absorbed and forms a gelatinous sealing layer, while the control groups (especially the blank and gauze groups) have continuous bleeding or loose blood clots. In summary, the 10% HAMA hemostatic sponge of the present application exhibits hemostatic efficiency far superior to traditional dressings and commercial products through the synergistic effect of liquid absorption, expansion and mechanical compression in the parenchymal organ trauma model, providing a more optimal solution for clinical acute trauma first aid.

[0073] Further, the biocompatibility of the hemostatic sponge can be characterized through experiments: configure Calcein-AM / PI double fluorescent dyes, wherein (1) dissolve Calcein-AM powder with anhydrous DMSO to prepare 1mM Calcein-AM stock solution; (2) dissolve PI powder with ultrapure water to prepare 1mg / mL PI stock solution. UV sterilize the hemostatic sponge, place it in a 96-well plate, inoculate NIH3T3 cells with a density of 2×10 5 mL -1 , and culture for 48h. Prepare a live and dead staining solution by taking 1μL Calcein-AM stock solution (1mM) + 1μL PI stock solution (1mg / mL) + 998μL preheated DPBS, remove the culture medium in the cell culture plate, gently rinse twice with preheated PBS, add 100μL staining working solution to each well to ensure coverage of the cell layer. Incubate in a 37℃ incubator for 20 minutes. Take pictures of the sample using a confocal microscope.

[0074] This invention characterizes the biocompatibility of hemostatic sponges using a Calcein-AM / PI dual-fluorescence staining method: NIH3T3 cells are seeded onto the surface of a 10% HAMA hemostatic sponge, cultured for 48 hours, and then stained with both live cells (Calcein-AM, green fluorescence) and dead cells (PI, red fluorescence). Figure 5 As can be seen, the Calcein channel exhibits dense green fluorescent dots, indicating that the viable cells are in good condition; the PI channel shows no obvious red signal, indicating that the proportion of dead cells is extremely low. This result stems from two mechanisms: firstly, methacryloyl hyaluronic acid (HAMA) is modified from natural hyaluronic acid and is inherently biocompatible. The hydrogel network formed by UV cross-linking and the porous structure constructed by subsequent drying provide a suitable microenvironment for cell adhesion and proliferation; secondly, the preparation process does not introduce any toxic residues (such as dialysis for impurity removal and aseptic treatment), avoiding chemical toxicity damage to cells. From the clinical requirements of medical hemostatic materials, biocompatibility is a core indicator for wound contact materials—the HAMA sponge of this invention needs to achieve rapid hemostasis while avoiding inflammatory reactions or cell necrosis. This experiment, through the visual characterization of live and dead cells, directly verifies its characteristics of no significant cytotoxicity and support for cell survival, providing a basis for the sponge's "safe contact with wound tissue" in trauma emergency care and ensuring the biosafety of surrounding tissues during hemostasis.

[0075] Furthermore, the in vivo biodegradability of the hemostatic sponge can be evaluated through the following experiments: Anesthesia: Tribromoethanol; intraperitoneal injection; mouse dose 0.2 ml / 10 g body weight, complete anesthesia within 5 minutes, maintenance anesthesia for approximately 10–40 minutes. If the operation time needs to be extended, 0.3 ml–0.5 ml of anesthetic can be injected intraperitoneally before the mouse awakens. Surgical area preparation: Fix the mouse on the operating table, remove the hair in the surgical area, and disinfect with povidone-iodine; remove iodine with alcohol. Preparation of the subcutaneous implantation area: Skin incision: Make skin incisions on both sides of the spine, approximately 0.5 cm long, with a distance of at least 1 cm between points on each side, two on each side; Separation of subcutaneous tissue and fascia: Bluntly dissect the subcutaneous tissue down to the superficial fascia muscle layer to create space for hydrogel implantation; use hemostatic forceps to separate the muscle at the implantation point. Hydrogel implantation: Implant the hydrogel sample into the pre-prepared subcutaneous cavity, ensuring no tension contact between the implant and surrounding tissue; In the blank control group, the skin was surgically incised, the subcutaneous tissue and fascia were separated, and the muscle at the implantation point was separated with hemostatic forceps. Incision Closure: Close the incision using appropriate suturing techniques, and suture the subcutaneous tissue and skin in layers. Postoperative Management: Disinfect the wound with povidone-iodine to prevent infection. Postoperative Observation: Observe mice at predetermined time points after implantation (e.g., 2, 4, 8, and 12 weeks) to assess the degradation of the hydrogel and the biological response of surrounding tissues. Sample Processing: Sacrifice new mice at 1, 3, and 5 weeks (and 7 weeks) postoperatively, and remove the muscles on both sides of the spine to observe the degradation rate at different time points after sponge implantation. Take muscle tissue 0.5–1.0 cm around the sample, fix with 10% formaldehyde, stain with hematoxylin and eosin, and observe the inflammatory response (grading), cell species and quantity characteristics, and capsule formation under an optical microscope.

[0076] This invention evaluates the in vivo biodegradability of a hemostatic sponge using a mouse subcutaneous implantation model: Mice were anesthetized with tribromoethanol, and a subcutaneous cavity was constructed via a standardized paraspinal surgery. A 10% HAMA hemostatic sponge was implanted, and samples were taken at 1, 3, and 5 weeks post-surgery. The muscle tissue surrounding the sponge was stained with hematoxylin and eosin (HE). Figure 6As can be seen, at 1 week, the sponge structure is relatively intact, and surrounding tissue begins to adhere to it; at 3 weeks, the sponge shrinks in volume, and new tissue invades the internal pores; at 5 weeks, the sponge further degrades, its structure becomes loose, and its integration with surrounding tissue increases. This degradation process stems from two mechanisms: first, methacrylamide hyaluronic acid (HAMA) retains the polysaccharide backbone of natural hyaluronic acid, which can be specifically enzymatically hydrolyzed by hyaluronidase in vivo, while the erosive effect of body fluids accelerates the disintegration of the network structure; second, the three-dimensional porous structure formed by ultraviolet cross-linking provides channels for tissue ingrowth, and the interaction between cells and matrix further promotes degradation. From the perspective of clinical needs analysis of medical hemostatic materials, controllable degradation is one of the core indicators: the material needs to gradually degrade after hemostasis to avoid secondary surgical removal (reducing the risk of trauma), and the degradation rate needs to match the tissue repair cycle (such as gradual degradation within 5 weeks, which matches the soft tissue repair process). In this experiment, the degradation process of the HAMA sponge and its histological morphology (no obvious inflammatory cell infiltration, consistent with...) are consistent. Figure 5 (Biocompatibility results) Collaborative verification: It possesses both the functional characteristics of "gradual metabolism after effective hemostasis" and ensures the biosafety of tissue repair.

[0077] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; phrases such as "preferred" or "according to a preferred embodiment" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the feature introduced by "preferred" is only an optional mode and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A method for preparing an expandable, high-efficiency hemostatic sponge, characterized in that, It includes the following steps: Preparation of HAMA: Double bonds are grafted onto the macromolecular backbone of hyaluronic acid by reacting hyaluronic acid with methacrylic anhydride. Preparation of HAMA hydrogel: A HAMA solution containing a photoinitiator is poured into a mold, and cross-linking is initiated by ultraviolet irradiation to obtain HAMA hydrogel; Remove the HAMA hydrogel from the mold and dry it to form a hemostatic sponge.

2. The preparation method according to claim 1, characterized in that, In the preparation of HAMA, hyaluronic acid is dissolved in a mixed solvent consisting of an organic solvent and water and stirred until completely dissolved. The solution is placed in an ice bath environment, and methacrylic anhydride is slowly added dropwise. The reaction is continued under ice bath conditions. After the reaction is completed, the pH of the system is adjusted by alkaline conditions, a precipitant is added to precipitate the product, and the crude HAMA product is obtained by centrifugation.

3. The preparation method according to claim 1 or 2, characterized in that, The crude HAMA product was dissolved and placed in a dialysis bag. Unreacted methacrylic anhydride and byproducts were removed by dialysis. After sterile filtration, the product was dried to obtain a solid HAMA sample.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The mixed solvent is a mixture of dimethylformamide and water; the precipitant is anhydrous ethanol, which is compatible with HAMA to efficiently separate the target product; after sterile filtration, a freeze-drying process is used to optimize the morphology and pore structure of HAMA.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The HAMA solid was redissolved in deionized water or phosphate buffer, a biocompatible photoinitiator was added, and the mixture was stirred until completely dissolved. The mixture was then injected into a mold. Crosslinking was initiated by irradiation with a UV light source. After crosslinking was completed, the hydrogel was removed from the mold and allowed to stand to promote the stabilization of the crosslinked network.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The concentration of HAMA in the mixture is 5% to 10%, more preferably 10%.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The biocompatible photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, or Irgacure 2959.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The drying process employs a two-step method: first, the hydrogel is dried in a constant temperature oven, and then the dried hydrogel is freeze-dried in a freeze dryer to preserve its internal porous structure.

9. The preparation method according to any one of claims 1 to 8, characterized in that, After the cross-linked hydrogel is removed from the mold, it is mechanically compressed before drying: the hydrogel is placed in a hydraulic device or mechanical pressure plate and a preset pressure is continuously applied. The pore structure is controlled by physical compression, so that the cross-linked network forms a denser elastic matrix, thereby increasing the volume expansion ratio when absorbing water.

10. An expandable, high-efficiency hemostatic sponge, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 9.

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