A hyaluronic acid / carboxymethyl chitosan hemostatic sponge and a preparation method thereof
The porous sponge formed by cross-linking oxidized hyaluronic acid with carboxymethyl chitosan solves the problems of slow water absorption and expansion and poor adhesion of existing hemostatic materials on deep and irregular wounds, achieving rapid and efficient hemostasis, and has good biocompatibility and complete degradability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST MEDICAL CENT CHINESE PLA GENERAL HOSPITAL
- Filing Date
- 2026-04-09
- Publication Date
- 2026-06-26
AI Technical Summary
Existing hemostatic materials have slow water absorption and expansion rates, insufficient expansion ratios, and difficulty in closely adhering to the wound surface when dealing with deep, irregular, and non-pressable wounds. They also pose risks of thermal damage, immunogenicity, and degradation mismatch.
A porous sponge was constructed by mixing oxidized hyaluronic acid and carboxymethyl chitosan and forming a hydrogel through a Schiff base reaction. The hydrogel was then cryogeled at low temperature and freeze-dried under vacuum. The reversible dynamic network structure was formed by cross-linking aldehyde and amino groups, which enabled rapid water absorption and expansion and efficient hemostasis.
The sponge expands more than three times its original size within seconds, achieving a synergistic effect of physical sealing and biochemical coagulation. It has good biocompatibility and is completely biodegradable, making it suitable for rapid hemostasis of deep and irregular wounds.
Smart Images
Figure CN122272872A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge and its preparation method. Background Technology
[0002] Traumatic bleeding, especially uncontrolled bleeding from deep, irregular, and non-compressible wounds, is one of the leading causes of death in clinical emergency care and battlefield rescue. Statistics show that more than half of trauma deaths worldwide occur before professional rescue arrives; therefore, developing hemostatic materials that can rapidly and effectively control bleeding is of significant clinical importance.
[0003] Currently, various hemostatic materials are used clinically, including gelatin sponges, oxidized cellulose sponges, zeolite-based hemostatic agents, and fibrin bandages. These materials can be effective in treating superficial, regular, and compressible bleeding wounds, but they often fail to achieve ideal hemostatic results when dealing with deep penetrating wounds, bleeding from narrow cavities, irregularly shaped wounds, and bleeding sites where external pressure cannot be applied.
[0004] Specifically, traditional hemostatic materials generally suffer from slow water absorption and expansion rates, resulting in insufficient expansion ratios. This prevents them from quickly and effectively adhering to irregular wound surfaces to form a physical seal, making them prone to detachment or displacement from the bleeding site. Consequently, hemostasis efficiency is low, and blood loss is excessive. Furthermore, some hemostatic materials, such as zeolite, release significant heat during water absorption, potentially causing thermal damage to surrounding healthy tissues. Some animal-derived protein-based hemostatic materials pose immunogenicity risks. Regarding tissue adhesion, most existing materials have weak bonding to the wound surface, making it difficult to maintain stable sealing under continuous bleeding or blood flow impact. In terms of degradation performance, many hemostatic materials are non-degradable in vivo, requiring secondary surgery for removal, increasing patient suffering and medical burden. While some materials are degradable, their degradation cycles do not match the tissue repair process; premature degradation may lead to secondary bleeding, while delayed degradation may trigger chronic inflammatory responses. Poor biocompatibility also limits the widespread application of some materials.
[0005] Therefore, developing a novel hemostatic material that can rapidly absorb water and expand to form a tight physical seal, possesses excellent tissue adhesion, and also has highly efficient procoagulant ability, good biocompatibility, and complete biodegradability in vivo, especially for the hemostatic needs of deep, irregular, and non-compressible bleeding wounds, remains an urgent technical problem to be solved in this field. Summary of the Invention
[0006] The purpose of this invention is to provide an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge and its preparation method. This hemostatic sponge does not require ultraviolet light, photoinitiators, or toxic crosslinking agents. It can form gel rapidly in situ at room temperature and has the characteristics of instant water absorption and expansion, high expansion ratio, strong tissue adhesion, efficient hemostasis, excellent biocompatibility, and complete biodegradability in vivo. It is suitable for rapid hemostasis of deep, irregular, and non-pressable bleeding wounds.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] The first aspect of this invention provides a method for preparing an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, characterized by comprising the following steps:
[0009] (1) Hyaluronic acid was dissolved in deionized water, and sodium periodate was added under light-protected conditions to carry out an oxidation reaction. After the reaction was completed, ethylene glycol was added to terminate the reaction. The oxidized hyaluronic acid was obtained by dialysis purification and freeze drying.
[0010] (2) Prepare oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively;
[0011] (3) Mix the oxidized hyaluronic acid solution with the carboxymethyl chitosan solution to allow the aldehyde group and the amino group to undergo a Schiff base reaction to form a hydrogel;
[0012] (4) The obtained hydrogel was cryogeled at low temperature and then freeze-dried under vacuum to obtain the hemostatic sponge.
[0013] Furthermore, in step (1), the molar ratio of hyaluronic acid repeating units to sodium periodate is 1:0.2 to 1:1.0.
[0014] Furthermore, the oxidation reaction in step (1) takes 2 to 6 hours.
[0015] Furthermore, the dialysis in step (1) uses a dialysis bag with a molecular weight cutoff of 3500~5000 Da, and the dialysis time is 3~5 days.
[0016] Further, in step (2), the mass concentration of the oxidized hyaluronic acid solution is 5wt%~15wt%, the mass concentration of the carboxymethyl chitosan solution is 1wt%~5wt%, and the solvent is deionized water or phosphate buffer solution with pH 7.2~7.4.
[0017] Further, in step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:1.
[0018] Furthermore, the temperature of the cryogel in step (4) is -30℃ to -5℃, and the cryogel time is not less than 48 hours.
[0019] Furthermore, the temperature of the vacuum freeze drying in step (4) is not higher than -40°C, and the drying time is not less than 48 hours.
[0020] Furthermore, the preparation method also includes the step of adding thrombin and / or antibiotics during the mixing process in step (3).
[0021] Adding thrombin and / or antibiotics during the mixing process in step (3) allows these bioactive molecules to be uniformly encapsulated within the three-dimensional network during hydrogel formation, further enhancing hemostasis efficiency and anti-infection capabilities. The active molecules are uniformly loaded, and no additional steps are required.
[0022] The hyaluronic acid molecular chain is selectively oxidized with sodium periodate to generate active aldehyde groups, while the carboxymethyl chitosan molecular chain retains amino groups. When the two are mixed in an aqueous phase, a Schiff base reaction occurs between the aldehyde and amino groups, forming dynamic imine bonds. These imine bonds act as cross-linking points, connecting the polymer chains into a three-dimensional network structure without the need for external cross-linking agents or ultraviolet light, and the reaction conditions are mild. Due to the reversibility of the imine bonds, the hydrogel can rearrange itself appropriately under stress, exhibiting a certain degree of flexibility. This reversibility also provides a molecular basis for subsequent in vivo degradation—hyaluronidase can degrade the hyaluronic acid backbone, lysozyme can degrade chitosan derivatives, and the degradation products can be safely metabolized after the cross-linked network is gradually destroyed. The above hydrogel is subjected to low-temperature cryogelation and vacuum freeze-drying, and the water is directly sublimated and removed in the form of ice crystals. The space originally occupied by water is transformed into an interconnected porous structure, resulting in a porous sponge that can be compressed and stored in a dry state. This structure gives the sponge an extremely high specific surface area and capillary force, allowing it to rapidly soak into and absorb large amounts of liquid within seconds of contact with blood or aqueous solutions. The dry pores quickly recover and expand after absorbing liquid, with a significant volume increase and an equilibrium expansion ratio of no less than 300%. When the sponge is placed in a deep or irregular bleeding wound, the water in the blood triggers rapid swelling of the sponge. The physical pressure generated by this volume expansion directly acts on the ruptured blood vessel, forming an immediate seal. Simultaneously, the sponge's porous surface can adsorb and activate platelets, locally concentrating coagulation factors and accelerating the coagulation cascade reaction. Thus, physical compression hemostasis and biochemical coagulation promotion achieve a synergistic effect.
[0023] A second aspect of the present invention provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, characterized in that it is prepared by the method described above in the present invention.
[0024] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:
[0025] 1. Rapid water absorption and expansion, with a high expansion ratio. The sponge has a three-dimensional interconnected porous structure, and its volume can expand more than 3 times within 30 seconds after contact with blood. The balanced expansion ratio is not less than 300%, which can form a tight physical compression seal on deep and irregular wounds, solving the problems of slow expansion and poor adhesion of traditional hemostatic materials.
[0026] 2. Significant hemostatic effect. While the sponge absorbs water and expands, its porous structure can quickly absorb water from the blood, concentrate platelets and coagulation factors, and activate platelets through the positive charge adsorption of carboxymethyl chitosan, accelerating the coagulation cascade reaction. The synergistic effect of physical compression and biochemical coagulation results in a significantly shorter hemostatic time than existing gelatin sponges or cellulose sponges.
[0027] 3. High biocompatibility. The raw material is a natural polysaccharide derivative, and the cross-linking process requires no external chemical cross-linking agents, no ultraviolet light, and no photoinitiators, avoiding the risk of toxic reagent residues. Cytotoxicity experiments have confirmed that the sponge has no significant toxicity to L929 cells and has good biocompatibility.
[0028] 4. Completely biodegradable in vivo. The hyaluronic acid in the sponge can be degraded by hyaluronidase in vivo, and the carboxymethyl chitosan can be degraded by lysozyme. The degradation cycle matches the tissue repair process, and the degradation products are safely metabolized, eliminating the need for a second surgery to remove them.
[0029] 5. The preparation process is simple. All operations are completed in an aqueous phase and at room temperature, requiring minimal equipment and using readily available raw materials, making it suitable for large-scale production and possessing promising prospects for clinical translation. Attached Figure Description
[0030] Figure 1 Example 1: Hemostatic sponge was used for hemostasis of ruptured liver in rats (① hemostatic sponge group; ② no treatment control group).
[0031] Figure 2 This is a graph showing the cytotoxicity detection of the hemostatic sponge Calcein-AM / PI in Example 1 (green: live cells; red: dead cells). Detailed Implementation
[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the embodiments are all commercially available products.
[0033] Example 1
[0034] This embodiment provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, the preparation method of which includes the following steps:
[0035] (1) Weigh 1.0g of hyaluronic acid and add it to 100mL of deionized water. Stir continuously for 8 hours until completely dissolved to obtain a homogeneous and transparent solution. Add 0.32g of sodium periodate under light-protected conditions and continue stirring at room temperature for 4 hours under light-protected conditions. After the reaction is completed, add 1mL of ethylene glycol and stir for 1 hour to terminate the oxidation reaction. Put the reaction mixture into a dialysis bag with a molecular weight cutoff of 3500Da and dialyze it with deionized water for 3 days, changing the deionized water 3 times a day to fully remove unreacted sodium periodate, ethylene glycol and small molecule byproducts. Pre-freeze the dialyzed solution at -80℃ for 12 hours and then transfer it to a vacuum freeze dryer to dry for 48 hours to obtain white and loose oxidized hyaluronic acid powder.
[0036] (2) Dissolve the above-mentioned oxidized hyaluronic acid powder in deionized water and stir until completely dissolved to prepare a 10wt% oxidized hyaluronic acid solution; dissolve carboxymethyl chitosan in deionized water and stir until completely dissolved to prepare a 3wt% carboxymethyl chitosan solution.
[0037] (3) Mix the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution rapidly at a volume ratio of 1:1, and vortex to mix them evenly to prepare the hydrogel pre-gel.
[0038] (4) Pour the obtained hydrogel pre-gel liquid into a cylindrical mold and freeze it in a -20℃ ultra-low temperature freezer for 3 days; then transfer it to a vacuum freeze dryer and dry it at -40℃ for 48 hours to obtain white, loose, porous, high expansion ratio OHA / CMCS hemostatic sponge.
[0039] Healthy adult SD rats, weighing approximately 250g, were anesthetized with isoflurane and fixed in a supine position. An incision was made along the midline of the abdomen to expose the liver. Two standard wounds of the same size were created on the liver surface: liver tissue was excised to form a defect of approximately 5mm × 5mm × 3mm, with continuous blood oozing from the wound. One wound was covered with a hemostatic sponge prepared in Example 1 and gently pressed for 10 seconds (hemostatic sponge group), while the other wound was left untreated (untreated control group). The hemostasis of the two wounds was observed and recorded. Figure 1 As shown, the wound in the hemostatic sponge group stopped bleeding quickly after the sponge was applied, and the sponge adhered tightly to the liver tissue; the wound in the untreated control group continued to bleed, and the hemostatic effect was significantly worse than that in the sponge group.
[0040] The hemostatic sponge sample from Example 1 was sterilized with ultraviolet light and then extracted according to the extraction standard for absorbent samples described in the national standard ISO 10993-5:2009. MEM complete culture medium was used as the extraction medium, and the extraction ratio of sample mass to extraction medium volume was 0.2 g / ml. Extraction was performed at 37°C for 24 hours to obtain the sponge extract. L929 cells were seeded in culture plates and cultured for 24 hours. The original culture medium was discarded, and the sponge extract was added for further culture for another 24 hours. Then, Calcein-AM (final concentration 2 μmol / L) and propidium iodide (final concentration 4.5 μmol / L) staining working solutions were added, and the cells were incubated at 37°C in the dark for 30 minutes. Under a fluorescence microscope, live cells showed green fluorescence, and dead cells showed red fluorescence. Figure 2 As shown, most cells in the field of view exhibit green fluorescence, with very little red fluorescence, indicating that the hemostatic sponge of Example 1 has no obvious cytotoxicity and has good biocompatibility.
[0041] Example 2
[0042] This embodiment provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, which differs from Example 1 in that, in step (2), a 5 wt% oxidized hyaluronic acid solution and a 5 wt% carboxymethyl chitosan solution are prepared; in step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:1, vortexed to ensure thorough mixing, and the system is directly placed at -5℃ for gelation for 3 days. The remaining steps are the same as in Example 1.
[0043] Example 3
[0044] This embodiment provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, which differs from Example 1 in that, in step (2), a 15wt% oxidized hyaluronic acid solution and a 1wt% carboxymethyl chitosan solution are prepared; in step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:1, vortexed to ensure thorough mixing, and the system is directly placed at -5℃ for gelation for 3 days. The remaining steps are the same as in Example 1.
[0045] Example 4
[0046] This embodiment provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, which differs from Example 1 in that, in step (2), a 5 wt% oxidized hyaluronic acid solution and a 1 wt% carboxymethyl chitosan solution are prepared; in step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:1, vortexed to ensure thorough mixing, and the system is directly placed at -5℃ for gelation for 3 days. The remaining steps are the same as in Example 1.
[0047] Example 5
[0048] This embodiment provides an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, which differs from Example 1 in that, in step (2), a 15wt% oxidized hyaluronic acid solution and a 5wt% carboxymethyl chitosan solution are prepared; in step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:1, vortexed to ensure thorough mixing, and the system is directly placed at -5℃ for gelation for 3 days. The remaining steps are the same as in Example 1.
[0049] Comparative Example 1
[0050] The difference between this comparative example and Example 1 is that in step (4), the obtained hydrogel pre-gel solution was placed at room temperature for 3 days to gel, and then transferred to a regular forced-air drying oven and dried at 50°C and normal pressure for 48 hours. The remaining steps are the same as in Example 1.
[0051] Performance testing
[0052] To verify the rapid water absorption and swelling performance of the hemostatic sponge of the present invention, we tested the volume expansion rate of Examples 1-5 and Comparative Example 1 at different time points. The test methods are as follows:
[0053] Take a sample of the hemostatic sponge to be tested (dry state), measure its radius and height with vernier calipers, calculate the initial volume (V0), and weigh the dry state mass using an analytical balance. Completely immerse the sample in phosphate-buffered saline (PBS, pH 7.4) at 37°C, and remove it after 10 seconds, 30 seconds, and 5 minutes. Gently blot away excess liquid from the surface with filter paper, and immediately measure the radius and height at these times to calculate the wet state volume (V0). t The volume expansion rate (%) is calculated using the following formula:
[0054] ;
[0055] Each sample was measured in triplicate, and the average value was taken.
[0056] The test results are shown in Table 1.
[0057] Table 1 Performance Test Results
[0058]
[0059] The performance test results above show that the hemostatic sponges prepared in Examples 1-5 of this invention all exhibit rapid water absorption and swelling characteristics. Their volume increases significantly within 10 seconds of contact with PBS, and the equilibrium swelling ratio reaches over 300% after 5 minutes. This indicates that the technical solution of this invention can obtain a highly expandable sponge that meets the requirements for rapid hemostasis. Comparative Example 1 did not undergo low-temperature cryogelation and vacuum freeze-drying; instead, it was dried directly in an oven after gelation at room temperature. The resulting material was a dense sheet rather than a porous sponge, and its volume hardly expanded after absorbing water. This demonstrates that low-temperature freezing combined with vacuum freeze-drying is a key process step in constructing a three-dimensional interconnected porous structure and achieving a high swelling ratio.
[0060] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing an oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, characterized in that, Includes the following steps: (1) Hyaluronic acid was dissolved in deionized water, and sodium periodate was added under light-protected conditions to carry out an oxidation reaction. After the reaction was completed, ethylene glycol was added to terminate the reaction. The oxidized hyaluronic acid was obtained by dialysis purification and freeze drying. (2) Prepare oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively; (3) Mix the oxidized hyaluronic acid solution with the carboxymethyl chitosan solution to allow the aldehyde group and the amino group to undergo a Schiff base reaction to form a hydrogel; (4) The obtained hydrogel was cryogeled at low temperature and then freeze-dried under vacuum to obtain the hemostatic sponge.
2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of hyaluronic acid repeating unit to sodium periodate is 1:0.2 to 1:1.
0.
3. The preparation method according to claim 1, characterized in that, The oxidation reaction in step (1) takes 2 to 6 hours.
4. The preparation method according to claim 1, characterized in that, The dialysis in step (1) uses a dialysis bag with a molecular weight cutoff of 3500~5000 Da, and the dialysis time is 3~5 days.
5. The preparation method according to claim 1, characterized in that, The mass concentration of the oxidized hyaluronic acid solution in step (2) is 5wt%~15wt%, the mass concentration of the carboxymethyl chitosan solution is 1wt%~5wt%, and the solvent is deionized water or phosphate buffer solution with pH 7.2~7.
4.
6. The preparation method according to claim 1, characterized in that, In step (3), the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are mixed at a volume ratio of 1:
1.
7. The preparation method according to claim 1, characterized in that, The temperature of the cryogel in step (4) is -30℃ to -5℃, and the cryogel time is not less than 48 hours.
8. The preparation method according to claim 1, characterized in that, The temperature of the vacuum freeze drying in step (4) shall not be higher than -40°C and the drying time shall not be less than 48 hours.
9. The preparation method according to claim 1, characterized in that, The preparation method further includes the step of adding thrombin and / or antibiotics during the mixing process in step (3).
10. An oxidized hyaluronic acid / carboxymethyl chitosan hemostatic sponge, characterized in that, It is prepared by the preparation method described in any one of claims 1-9.