A biological protein polyurethane composite hemostatic stent and a preparation method thereof

Abstract

This invention discloses a bio-protein polyurethane composite hemostatic stent and its preparation method, belonging to the field of medical devices. The hemostatic stent comprises, by weight, 10-50 parts bio-protein, 50-90 parts polyurethane, 0.1-0.5 parts crosslinking agent, and 2-5 parts pore-forming agent. This invention, by combining bio-protein and polyurethane to form a stable interpenetrating network structure, produces a hemostatic stent with good mechanical properties, a controllable degradation rate, and a porous structure. It can closely adhere to the uterine cavity wound, achieving rapid hemostasis and long-term anti-adhesion. It is also safely degraded and absorbed by the human body, eliminating the need for secondary removal. Furthermore, it can load bioactive excipients, effectively promoting wound healing.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and in particular to a bio-protein polyurethane composite hemostatic stent and its preparation method. Background Technology

[0002] Post-hysteroscopic adhesions refer to the adhesion of the endometrial lining of the uterine wall to each other after surgery within the uterine cavity, forming fibrous tissue. Any surgery that damages the endometrium can cause adhesions, especially procedures requiring tissue removal or extensive curettage, such as induced abortion or dilation and curettage (D&C), uterine septum resection, submucosal myomectomy, and the hysteroscopic adhesiolysis itself (surgeries treating adhesions can also lead to new adhesions). Intrauterine adhesions can cause menstrual abnormalities, infertility, or recurrent miscarriages. Some patients may experience cyclical lower abdominal pain, especially when menstrual flow is obstructed.

[0003] The current main solution to this problem is prevention, which involves doctors placing a temporary balloon stent or injecting an anti-adhesion gel into the uterine cavity after surgery to physically isolate the uterine wall and buy time for endometrial repair. However, due to its large size, the balloon stent may cause discomfort such as lower abdominal distension, backache, and uterine contraction pain. Furthermore, the catheter of the balloon extends from the uterine cavity through the cervix and vagina to the outside of the body, disrupting the natural barrier function of the cervix and providing a potential pathway for ascending bacterial infection, increasing the risk of endometritis. In addition, the balloon stent needs to be removed a second time after 5-7 days, which may cause secondary harm to the patient.

[0004] The main component of commonly used anti-adhesion gels is sodium hyaluronate. These gels are expensive, increasing the financial burden on patients. Due to their inherent properties, gels are very easy to be excreted with the blood, which reduces their coverage and effectiveness on the wound surface. As a result, the gel may be completely drained before the wound has healed, and the effect varies from person to person and is not stable.

[0005] Therefore, there is an urgent need for a composite hemostatic stent that is long-lasting, anti-adhesion, biodegradable, does not require secondary removal, and promotes wound repair. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-protein polyurethane composite hemostatic stent to solve the problems of adhesion after hysteroscopy in the prior art, the need for secondary removal of existing stents, which can easily cause patient discomfort, bacterial infection, or easy loss of anti-adhesion gel and unstable effect.

[0007] The present invention also aims to provide a preparation method for preparing a composite hemostatic stent with controllable mechanical properties, degradability, and the ability to accelerate wound healing while preventing wound adhesion.

[0008] In a first aspect, the present invention provides a bioprotein polyurethane composite hemostatic stent, comprising, by weight, 10-50 parts bioprotein, 50-90 parts polyurethane, 0.1-0.5 parts crosslinking agent, and 2-5 parts pore-forming agent.

[0009] By adopting the above technical solutions, this invention achieves synergistic optimization of hemostatic stent materials in terms of mechanical properties, biocompatibility, and functional activity through the composite design of biological protein and polyurethane, effectively solving the technical problems of secondary trauma risk, unstable effect, and single function of existing intrauterine anti-adhesion products.

[0010] This invention utilizes biological proteins, preferably modified silk fibroin. Maleic anhydride carboxylation significantly enhances the water solubility and reactivity of the silk fibroin, allowing the introduced carboxyl groups on its molecular chain to form hydrogen bonds with the isocyanate groups or hydroxyl groups at the ends of the polyurethane molecular chain, constructing a composite system with a semi-interpenetrating network structure. Simultaneously, the polycaprolactone diol and polyethylene glycol segments in the polyurethane soft segments impart good flexibility to the material, while the lysine diisocyanate in the hard segments provides degradable urea bond connection points, enabling the composite material to achieve controlled degradation while maintaining appropriate mechanical support strength.

[0011] Crosslinking agents, such as genipin, have active groups in their molecules that can crosslink with the amino groups of silk fibroin to form stable complexes. This allows for in-situ crosslinking and curing under mild conditions, avoiding the cytotoxicity issues associated with traditional chemical crosslinking agents. Pore-forming agents, such as mannitol, create a continuous porous structure within the scaffold through a freeze-crystallization sublimation mechanism, providing pathways for cell migration, nutrient delivery, and metabolic waste removal. For example, the addition of ginsenoside Rg1 and vitamin E succinate can further enhance the material's antioxidant capacity and angiogenesis-promoting activity, accelerating the wound healing process.

[0012] Preferably, the biological protein includes one or more of the following: modified silk fibroin, silk protein, mussel protein, recombinant collagen, and human thrombin.

[0013] Preferably, the raw materials for polyurethane include polyols, diisocyanates, and catalysts in a mass ratio of 1:(0.3-1.2):(0.001-0.005).

[0014] Preferably, polyurethane is prepared by the following method: Polyols are placed in a reaction vessel and dried to remove water. Diisocyanate and catalyst are added, and the reaction is carried out at 60-80°C under an inert gas for 3-5 hours. The temperature is then lowered to 40-50°C and anhydrous ethanol is added to terminate the reaction. The product is then washed and dried to obtain polyurethane.

[0015] Preferably, polyols include polycaprolactone diol and polyethylene glycol.

[0016] Preferably, the diisocyanate includes lysine diisocyanate and hexamethylene diisocyanate.

[0017] Preferably, the catalyst comprises dibutyltin dilaurate.

[0018] Preferably, the modified silk fibroin is prepared by the following method: Silk fibroin is dissolved in a solvent, maleic anhydride is added and reacted at 70-100℃. After the reaction is completed, the carboxylated silk fibroin is obtained by dialysis. The carboxylated silk fibroin is then dissolved in a calcium alcohol water ternary solution, dialyzed, and dried to obtain the final product. The mass ratio of silk fibroin to maleic anhydride is 1:(1-6).

[0019] Preferably, the crosslinking agent includes genipin.

[0020] Preferably, the pore-forming agent includes mannitol.

[0021] Preferably, by weight, it also includes 0.1 to 0.5 parts of bioactive excipients.

[0022] Preferably, the bioactive excipients include ginsenoside Rg1 and vitamin E succinate.

[0023] Secondly, the present invention also provides a preparation method, comprising the following steps: S1. Prepare a biological protein solution, stir and degas it, then pour it into a uterine cavity matching mold, and freeze-dry it to obtain a biological protein scaffold. S2. Prepare a polyurethane solution, add the remaining components, stir and degas, immerse the biological protein scaffold in the polyurethane solution, and freeze-dry to obtain the scaffold preform. S3. Place the prefabricated stent in an environment of 20-40℃ and 75%-100% relative humidity, and allow it to crosslink and cure in situ for 12-18 hours to obtain a bioprotein polyurethane composite hemostatic stent.

[0024] Preferably, in step S2, freeze drying includes pre-freezing at 4°C for 1-2 hours, freezing at -20°C for 2-3 hours, and freezing at -55°C for 6-8 hours.

[0025] Preferably, the solvent for the polyurethane solution is 1,4-dioxane.

[0026] The beneficial effects of this invention are: This invention utilizes a biocompatible protein scaffold framework to provide physical support for damaged endometrium and prevent adhesions caused by contact between uterine cavity walls. Furthermore, polyurethane is introduced to reinforce the biocompatible protein scaffold. Polyurethane possesses excellent mechanical properties, endowing the scaffold with suitable flexibility and shape memory, allowing it to closely conform to the uterine cavity shape and avoiding discomfort such as lower abdominal distension and backache caused by excessive volume. By combining polyurethane with biocompatible protein, a cross-linking agent induces in-situ cross-linking reactions between the polyurethane and biocompatible protein molecular chains, forming a stable interpenetrating network structure. This network structure effectively regulates the degradation rate of the scaffold in vivo. After fulfilling its anti-adhesion function, it can degrade into non-toxic amino acids and small molecules that can be safely absorbed or metabolized by the body, completely avoiding the trauma and infection risks associated with a secondary removal surgery. Simultaneously, this invention preferably uses modified silk fibroin, introducing carboxyl groups through maleic anhydride and treating it with a calcium alcohol-water ternary solution, significantly improving the water solubility of silk fibroin and its cross-linking reactivity with polyurethane, ensuring the uniformity and stability of the composite scaffold structure. Furthermore, the scaffold prepared by this invention has a porous internal structure, which can not only efficiently absorb wound exudate and activate the intrinsic coagulation mechanism to achieve rapid hemostasis, but also load bioactive excipients such as ginsenoside Rg1 and vitamin E succinate to continuously release them to promote endometrial cell proliferation and angiogenesis, accelerate wound repair, and block adhesion formation. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0028] A bioprotein-polyurethane composite hemostatic stent, by weight, comprises 10-50 parts bioprotein, 50-90 parts polyurethane, 0.1-0.5 parts crosslinking agent, and 2-5 parts pore-forming agent.

[0029] By adopting the above technical solutions, this invention achieves synergistic optimization of hemostatic stent materials in terms of mechanical properties, biocompatibility, and functional activity through the composite design of biological protein and polyurethane, effectively solving the technical problems of secondary trauma risk, unstable effect, and single function of existing intrauterine anti-adhesion products.

[0030] This invention utilizes biological proteins, preferably modified silk fibroin. Maleic anhydride carboxylation significantly enhances the water solubility and reactivity of the silk fibroin, allowing the introduced carboxyl groups on its molecular chain to form hydrogen bonds with the isocyanate groups or hydroxyl groups at the ends of the polyurethane molecular chain, constructing a composite system with a semi-interpenetrating network structure. Simultaneously, the polycaprolactone diol and polyethylene glycol segments in the polyurethane soft segments impart good flexibility to the material, while the lysine diisocyanate in the hard segments provides degradable urea bond connection points, enabling the composite material to achieve controlled degradation while maintaining appropriate mechanical support strength.

[0031] Crosslinking agents, such as genipin, have active groups in their molecules that can crosslink with the amino groups of silk fibroin to form stable complexes. This allows for in-situ crosslinking and curing under mild conditions, avoiding the cytotoxicity issues associated with traditional chemical crosslinking agents. Pore-forming agents, such as mannitol, create a continuous porous structure within the scaffold through a freeze-crystallization sublimation mechanism, providing pathways for cell migration, nutrient delivery, and metabolic waste removal. For example, the addition of ginsenoside Rg1 and vitamin E succinate can further enhance the material's antioxidant capacity and angiogenesis-promoting activity, accelerating the wound healing process.

[0032] In some embodiments, the biological protein includes one or more of modified silk fibroin, silk fibroin, mussel protein, recombinant collagen, and human thrombin.

[0033] By adopting the above technical solutions, the various biological proteins selected in this approach all possess excellent biocompatibility and adaptability to intrauterine repair scenarios, allowing for flexible combinations and synergistic effects based on clinical needs. Specifically, silk fibroin and recombinant collagen contain RGD cell adhesion sequences, which can specifically promote the adhesion and proliferation of endometrial epithelial cells and stromal cells, providing stable bioactive support for endometrial wound repair; mussel protein is rich in dopa functional groups, which can significantly enhance the adhesion of the stent to the wound in the moist environment of the intrauterine cavity, preventing stent displacement and subsequent adhesion failure; human thrombin can directly activate the coagulation cascade reaction, significantly shortening wound clotting time and enhancing the intraoperative and postoperative hemostatic effects of the stent.

[0034] In some embodiments, the raw materials for polyurethane include polyols, diisocyanates, and catalysts in a mass ratio of 1:(0.3-1.2):(0.001-0.005).

[0035] Furthermore, polyurethane is prepared by the following method: Polyols are placed in a reaction vessel and dried to remove water. Diisocyanate and catalyst are added, and the reaction is carried out at 60-80°C under an inert gas for 3-5 hours. The temperature is then lowered to 40-50°C and anhydrous ethanol is added to terminate the reaction. The product is then washed and dried to obtain polyurethane.

[0036] Furthermore, polyols include polycaprolactone diol and polyethylene glycol.

[0037] Furthermore, diisocyanates include lysine diisocyanate and hexamethylene diisocyanate.

[0038] Furthermore, the catalyst includes dibutyltin dilaurate.

[0039] By employing the above technical solutions, biodegradable polyurethane materials are prepared using polyols and diisocyanates. Polycaprolactone diol provides hydrophobic soft segments for the polyurethane, precisely controlling the degradation rate and mechanical strength of the material. Polyethylene glycol provides hydrophilic soft segments, enhancing the material's hydrophilicity and blood adsorption capacity. The combination of these two components allows for wide-range control of the polyurethane's hydrophilicity / hydrophobicity, flexibility, and degradation cycle. The selected lysine diisocyanate and hexamethylene diisocyanate are both aliphatic diisocyanates with excellent biocompatibility. The degradation products of lysine diisocyanate are essential amino acids for the human body, further enhancing the material's biosafety. The polyurethane prepared through this polymerization process has numerous ester, hydroxyl, and amino groups on its molecular chain that can form hydrogen bonds with carboxylated biological proteins. This allows for the construction of a stable interpenetrating network structure with biological proteins, achieving a synergistic match between mechanical properties and degradation behavior, thus overcoming the phase separation defects of simple protein-polyurethane blends.

[0040] In some embodiments, modified silk fibroin is prepared by the following method: Silk fibroin is dissolved in a solvent, maleic anhydride is added and reacted at 70-100℃. After the reaction is completed, the carboxylated silk fibroin is obtained by dialysis. The carboxylated silk fibroin is then dissolved in a calcium alcohol water ternary solution, dialyzed, and dried to obtain the final product. Furthermore, the mass ratio of silk fibroin to maleic anhydride is 1:(1-6).

[0041] By employing the above technical solutions, maleic anhydride is used to modify silk fibroin, introducing a large number of carboxyl groups onto the silk fibroin molecular chain. By adjusting the mass ratio of silk fibroin to maleic anhydride from 1:1 to 1:6, the water solubility of silk fibroin can be improved, enabling it to form a high-concentration homogeneous solution in room temperature water. More importantly, the introduced carboxyl groups can form strong hydrogen bonds with the polyurethane molecular chain, while providing more reaction sites for the cross-linking agent genipin, significantly improving the interfacial compatibility and cross-linking network stability of the composite system. Subsequent secondary treatment of the carboxylated silk fibroin with a calcium alcohol-water ternary solution eliminates intramolecular aggregation of the carboxylated silk fibroin, further improving its water solubility and molecular chain flexibility, fully exposing the active sites, ensuring the homogeneity of the subsequent cross-linking reaction and the composite system, and avoiding problems such as uneven performance and excessively rapid local degradation within the scaffold.

[0042] In some embodiments, the crosslinking agent includes genipin.

[0043] By adopting the above technical solutions, this solution selects genipin, a natural iridoid compound, as a cross-linking agent. Genipin possesses excellent biocompatibility, exhibiting no cytotoxicity, no sensitization, and no risk of residual effects in vivo, fully meeting the safety requirements for Class III intrauterine implantable medical devices. The active groups of genipin can undergo specific nucleophilic addition reactions with free amino groups on biological protein molecular chains, forming a stable dihydropyran ring covalent cross-linked structure in a mild, ambient, aqueous environment. This process requires no strong acid or base catalysis and does not damage the bioactivity of the biological protein or the molecular structure of the polyurethane. Simultaneously, the cross-linking reaction rate of genipin is gradual and controllable, allowing for precise regulation of the cross-linking density of the composite system through the amount added, reaction temperature, and time. This enables a wide range of control over the mechanical properties, swelling rate, and degradation cycle of the stent. In the interpenetrating network system of this invention, the three-dimensional protein network formed by genipin cross-linking intertwines and synergistically reinforces the physical cross-linked network of polyurethane, allowing the stent to maintain structural integrity for a long time in the moist environment of the uterine cavity, preventing swelling, collapse, and interlayer separation, thus providing stable structural support for long-term anti-adhesion.

[0044] In some embodiments, the pore-forming agent includes mannitol.

[0045] By adopting the above technical solutions, this scheme selects mannitol as a pore-forming agent to regulate the porous structure inside the scaffold. Mannitol is a polyhydroxy water-soluble compound that can act as a heterogeneous ice crystal nucleating agent during freezing, significantly reducing the nucleation barrier of water molecules and generating a large number of uniform ice crystal nucleation sites. Simultaneously, it can adsorb at the ice crystal growth interface, limiting the directional excessive growth of ice crystals. The pore structure regulated by mannitol can, on the one hand, rapidly adsorb exudate and blood from the uterine cavity wound, enriching blood cells and coagulation factors to achieve rapid hemostasis; on the other hand, it can provide a three-dimensional channel for the infiltration and migration of endometrial cells, while ensuring the transport of nutrients and metabolic waste, accelerating the physiological repair of the endometrial wound.

[0046] In some embodiments, the mixture may further include 0.1 to 0.5 parts by weight of bioactive excipients.

[0047] Furthermore, the bioactive excipients include ginsenoside Rg1 and vitamin E succinate.

[0048] By adopting the above technical solutions, this approach, through the addition of low-dose bioactive excipients, further enhances the anti-inflammatory and repair-promoting effects of the stent without affecting its main structure and performance, achieving a synergistic effect of physical anti-adhesion and biological repair promotion. Specifically, ginsenoside Rg1 can inhibit the NF-κB inflammatory signaling pathway, reduce the release of inflammatory factors in the intrauterine wound, decrease macrophage infiltration and scar tissue formation, and simultaneously promote endometrial cell proliferation and angiogenesis, accelerating the physiological healing of the wound. Vitamin E succinate possesses strong antioxidant activity, can scavenge oxygen free radicals in the wound area, reduce oxidative stress damage to endometrial cells, and synergistically work with ginsenoside Rg1 to exert an anti-inflammatory effect, further reducing the risk of postoperative adhesion recurrence.

[0049] A preparation method comprising the following steps: S1. Prepare a biological protein solution, stir and degas it, then pour it into a uterine cavity matching mold, and freeze-dry it to obtain a biological protein scaffold. S2. Prepare a polyurethane solution, add the remaining components, stir and degas, immerse the biological protein scaffold in the polyurethane solution, and freeze-dry to obtain the scaffold preform. S3. Place the prefabricated stent in an environment of 20-40℃ and 75%-100% relative humidity, and allow it to crosslink and cure in situ for 12-18 hours to obtain a bioprotein polyurethane composite hemostatic stent.

[0050] By adopting the above technical solutions, in step S1, a three-dimensional porous scaffold of biological protein is first prepared by freeze-drying, which constructs a continuous bioactive framework for the composite system, ensuring the overall biocompatibility and repair-promoting function of the scaffold. In step S2, polyurethane solution is uniformly infiltrated into the pores of the biological protein scaffold by impregnation, and then freeze-dried a second time, so that polyurethane forms a uniform coating reinforcement layer on the surface of the protein framework. The two form an interpenetrating network structure, realizing the molecular-level composite of protein bioactivity and polyurethane mechanical properties without macroscopic phase separation. In step S3, constant temperature and humidity in-situ crosslinking and curing allows the crosslinking reaction to proceed slowly in a mild hydration environment, resulting in a more uniform distribution of the crosslinking network and better structural stability and mechanical properties of the scaffold.

[0051] In some embodiments, in step S2, freeze drying includes pre-freezing at 4°C for 1-2 hours, freezing at -20°C for 2-3 hours, and freezing at -55°C for 6-8 hours.

[0052] By adopting the above technical solutions, this solution uses a gradient cooling freezing procedure to control the nucleation and growth process of ice crystals in the pores of the polyurethane solution within the biological protein scaffold, ensuring the porous structure of the composite scaffold.

[0053] In some embodiments, the solvent for the polyurethane solution is 1,4-dioxane.

[0054] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0055] Preparation Example Preparation Example 1-1: A modified silk fibroin was prepared by the following method: 0.5 g of silk fibroin was dissolved in 3.125 g of molten 1-butyl-3-methylimidazolium chloride, 10 mL of anhydrous dimethyl sulfoxide was added, and then 1.5 g of maleic anhydride was added to carry out the reaction. The reaction was carried out at 80 °C for 3 h. After the reaction was completed, the reaction solution was first dialyzed with water to obtain insoluble carboxylated silk fibroin. The insoluble carboxylated silk fibroin was then dissolved in a calcium alcohol water ternary solution with a molar ratio of CaCl2-EtOH-H2O of 1:2:8. The solution was dialyzed again with ultrapure water, and the dialysate was freeze-dried to obtain water-soluble carboxylated silk fibroin.

[0056] Preparation Example 2-1: A polyurethane was prepared by the following method: 50g of polycaprolactone diol and 50g of polyethylene glycol were placed in a reaction vessel, heated to 80℃, and stirred until homogeneous. The mixture was then dehydrated under vacuum for 1 hour. The temperature was lowered to 60℃, and 25g of lysine diisocyanate and 0.5g of dibutyltin dilaurate catalyst were added. The reaction was carried out under nitrogen protection for 4 hours. After the reaction was completed, the temperature was lowered to 40℃, and anhydrous ethanol was added to terminate the reaction. The product was precipitated with deionized water, washed three times, and dried under vacuum at 40℃ to obtain a polyurethane.

[0057] Example Example 1: A bio-protein polyurethane composite hemostatic stent was prepared by the following method: S1. Weigh 30 parts of the modified silk fibroin obtained in Preparation Example 1-1, add it to ultrapure water, stir and dissolve it at room temperature to prepare a solution with a mass concentration of 5%, and continue stirring for 2 hours to allow it to fully swell and disperse. After degassing, slowly pour the solution into a sterilized uterine cavity adaptable sheet mold. The mold material is medical-grade polytetrafluoroethylene. Place the poured mold in a freeze dryer, first lower it to -20°C at a cooling rate of 1°C / min, keep it for 4 hours to allow the solution to freeze completely, then lower it to -55°C, evacuate to 0.08 mbar, freeze dry for 24 hours to obtain a biological protein scaffold.

[0058] S2. Weigh 70 parts of the polyurethane prepared in Preparation Example 2-1, add it to 1,4-dioxane, and heat and stir in a water bath at 50°C to dissolve it and prepare a solution with a mass concentration of 8%. After the polyurethane is completely dissolved, add 0.3 parts of genipin, 3.5 parts of mannitol, 0.2 parts of ginsenoside Rg1, and 0.1 parts of vitamin E succinate in sequence, and continue stirring for 1 hour to make the components evenly dispersed. After degassing, completely immerse the biological protein scaffold prepared in S1 in the above polyurethane mixed solution to allow the solution to fully penetrate the scaffold pores. Place the impregnated scaffold back in a freeze dryer, first cool it to 4°C at 2°C / min and pre-freeze it for 1.5 hours; then cool it to -20°C at 3°C / min and freeze it for 2.5 hours; finally cool it to -55°C, evacuate it to 0.08 mbar, and freeze-dry it for 7 hours to obtain the scaffold preform.

[0059] S3. Transfer the prefabricated stent to a constant temperature and humidity incubator, set the temperature to 30℃ and the relative humidity to 87%, and allow it to stand for cross-linking and curing for 15 hours to obtain a biological protein polyurethane composite hemostatic stent.

[0060] Examples 2 to 5 describe a bio-protein polyurethane composite hemostatic stent, differing from Example 1 only in the adjustment of the raw material ratio of the composite hemostatic stent, as shown in Table 1: Table 1. Raw material list for Examples 1 to 5

[0061] Comparative Example Comparative Example 1: A biological protein scaffold was prepared by the following method: S1. Weigh 30 parts of the modified silk fibroin prepared in Preparation Example 1-1, add it to ultrapure water, and then add 0.3 parts of genipin, 3.5 parts of mannitol, 0.2 parts of ginsenoside Rg1 and 0.1 parts of vitamin E succinate in sequence. Stir and dissolve at room temperature to prepare a solution with a mass concentration of 5%, and continue stirring for 2 hours to allow it to fully swell and disperse. After degassing, slowly pour the solution into a sterilized uterine cavity adaptable sheet mold. The mold material is medical-grade polytetrafluoroethylene. Place the poured mold in a freeze dryer, first lower the temperature to -20°C at a cooling rate of 1°C / min, keep it for 4 hours to allow the solution to freeze completely, then lower the temperature to -55°C, evacuate to 0.08 mbar, and freeze dry for 24 hours to obtain a biological protein scaffold. S2. Transfer the biological protein scaffold to a constant temperature and humidity incubator, set the temperature to 30℃ and the relative humidity to 87%, and allow it to stand for 15 hours to cross-link and solidify, thus obtaining a biological protein scaffold.

[0062] Comparative Example 2, a biological protein scaffold, was prepared by the following method: S1. Weigh 30 parts of the modified silk fibroin obtained in Preparation Example 1-1, add it to ultrapure water, stir and dissolve it at room temperature to prepare a solution with a mass concentration of 5%, and continue stirring for 2 hours to allow it to fully swell and disperse. After degassing, slowly pour the solution into a sterilized uterine cavity adaptable sheet mold. The mold material is medical-grade polytetrafluoroethylene. Place the poured mold in a freeze dryer, first lower it to -20°C at a cooling rate of 1°C / min, keep it for 4 hours to allow the solution to freeze completely, then lower it to -55°C, evacuate to 0.08 mbar, freeze dry for 24 hours to obtain a biological protein scaffold.

[0063] S2. Weigh 70 parts of the polyurethane prepared in Preparation Example 2-1, add it to 1,4-dioxane, and heat and stir in a water bath at 50°C to dissolve it and prepare a solution with a mass concentration of 8%. After the polyurethane is completely dissolved, add 3.5 parts of mannitol, 0.2 parts of ginsenoside Rg1, and 0.1 parts of vitamin E succinate in sequence, and continue stirring for 1 hour to make the components evenly dispersed. After degassing, completely immerse the biological protein scaffold prepared in S1 in the above polyurethane mixed solution to allow the solution to fully penetrate the scaffold pores. Place the impregnated scaffold back in a freeze dryer, first cool it to 4°C at 2°C / min and pre-freeze it for 1.5 hours; then cool it to -20°C at 3°C / min and freeze it for 2.5 hours; finally cool it to -55°C, evacuate it to 0.08 mbar, and freeze-dry it for 7 hours to obtain a biological protein scaffold.

[0064] Performance testing: 1. In vitro degradation test: Prepare simulated uterine cavity fluid (pH 7.4 PBS, containing appropriate amounts of lysozyme and collagenase), immerse the stents of Examples 1 to 5 and Comparative Examples 1 to 2 in it, shake on a shaker at 37°C, remove periodically, absorb surface moisture, weigh, and calculate the weight residual rate; 2. Cell compatibility testing: Referring to GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests", the MTT assay was used to detect the cytotoxicity of the scaffold extract against mouse fibroblasts (L929). Scaffold samples were immersed in serum-free culture for 24 h to prepare the extract. L929 cells were seeded in 96-well plates and cultured for 24 h. After discarding the original culture medium, the scaffold extract was added, and the cells were cultured for another 24 h and 48 h. MTT solution was then added, and after incubation for 4 h, DMSO was added to dissolve formazan crystals. The absorbance was measured at 490 nm, and the relative cell proliferation rate was calculated. 3. Animal experiments and model establishment: SD female rats were selected, and a uterine adhesion model was established by curettage. The rats were randomly divided into three groups: a sham surgery group, a stent injection group (stents from the example and control group were implanted after curettage), and a control group. Four weeks later, the animals were sacrificed, and the uterus was harvested for immunohistochemical detection of CD68 (macrophage marker) expression.

[0065] Table 2 Performance Test Results

[0066] As shown in Table 2, by constructing an interpenetrating network structure of biological protein and polyurethane, the 4-week residual rate of Example 1 was significantly higher than that of Comparative Example 1, which used a pure protein scaffold, demonstrating that the introduction of polyurethane effectively regulated the degradation cycle. Meanwhile, Example 1 exhibited the best cell compatibility, and the number of CD68-positive cells was significantly lower than that of Comparative Example 1 and the model control group, indicating that the scaffold provided by this invention can reduce inflammatory responses. Its mechanism of action lies in the following: the active sites provided by the modified silk fibroin form stable cross-links with the polyurethane, preventing burst release and heterogeneous degradation of the material; while the sustained release of the bioactive excipients ginsenoside Rg1 and vitamin E succinate further exerts a synergistic effect of anti-inflammatory and repair-promoting properties, thereby accelerating wound healing while preventing adhesion.

[0067] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A bio-protein polyurethane composite hemostatic stent, characterized in that, By weight, it includes 10-50 parts of biological protein, 50-90 parts of polyurethane, 0.1-0.5 parts of crosslinking agent, and 2-5 parts of pore-forming agent.

2. The bio-protein polyurethane composite hemostatic stent according to claim 1, characterized in that, The biological protein includes one or more of the following: modified silk fibroin, silk protein, mussel protein, recombinant collagen, and human thrombin.

3. The bio-protein polyurethane composite hemostatic stent according to claim 1, characterized in that, The raw materials for the polyurethane include polyols, diisocyanates, and catalysts in a mass ratio of 1:(0.3-1.2):(0.001-0.005).

4. The bio-protein polyurethane composite hemostatic stent according to claim 3, characterized in that, The polyurethane is prepared by the following method: Polyols are placed in a reaction vessel and dried to remove water. Diisocyanate and catalyst are added, and the reaction is carried out at 60-80°C under an inert gas for 3-5 hours. The temperature is then lowered to 40-50°C and anhydrous ethanol is added to terminate the reaction. The product is then washed and dried to obtain polyurethane.

5. The bio-protein polyurethane composite hemostatic stent according to claim 3, characterized in that, The polyols include polycaprolactone diol and polyethylene glycol; The diisocyanate includes lysine diisocyanate and hexamethylene diisocyanate; The catalyst includes dibutyltin dilaurate.

6. The bio-protein polyurethane composite hemostatic stent according to claim 1, characterized in that, The modified silk fibroin was prepared by the following method: Silk fibroin is dissolved in a solvent, maleic anhydride is added and reacted at 70-100℃. After the reaction is completed, the carboxylated silk fibroin is obtained by dialysis. The carboxylated silk fibroin is then dissolved in a calcium alcohol water ternary solution, dialyzed, and dried to obtain the final product. The mass ratio of silk fibroin to maleic anhydride is 1:(1-6).

7. The bio-protein polyurethane composite hemostatic stent according to claim 6, characterized in that, The crosslinking agent includes genipin; The pore-forming agent includes mannitol.

8. The bio-protein polyurethane composite hemostatic stent according to claim 1, characterized in that, It also includes 0.1 to 0.5 parts by weight of bioactive excipients; The bioactive excipients include ginsenoside Rg1 and vitamin E succinate.

9. A preparation method for preparing the bio-protein polyurethane composite hemostatic stent according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Prepare a biological protein solution, stir and degas it, then pour it into a uterine cavity matching mold, and freeze-dry it to obtain a biological protein scaffold. S2. Prepare a polyurethane solution, add the remaining components, stir and degas, immerse the biological protein scaffold in the polyurethane solution, and freeze-dry to obtain the scaffold preform. S3. Place the prefabricated stent in an environment of 20-40℃ and 75%-100% relative humidity, and allow it to crosslink and cure in situ for 12-18 hours to obtain a bioprotein polyurethane composite hemostatic stent.

10. The preparation method according to claim 9, characterized in that, In step S2, the freeze-drying includes pre-freezing at 4°C for 1-2 hours, freezing at -20°C for 2-3 hours, and freezing at -55°C for 6-8 hours.

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