Preparation of low-swelling medical tissue sealant and application of low-swelling medical tissue sealant in dura mater repair

Abstract

The invention belongs to the technical field of biomedical materials, and discloses preparation of a low-swelling medical tissue sealant and application of the low-swelling medical tissue sealant in dura mater repair. The invention discloses a sealant. The sealant is prepared from the following raw materials: multi-arm polyethylene glycol active ester and aminated gelatin. The invention provides a sealant. The sealant is formed by crosslinking multi-arm polyethylene glycol active ester and aminated gelatin through amido bonds. The sealant has the advantages of rapid gelling, high mechanical strength, strong tissue adhesion, ultralow swelling ratio, controllable degradation and excellent biocompatibility. The sealant can realize in-situ sealing and repairing of dura mater injury and prevent leakage of cerebrospinal fluid, has antibacterial, anti-infection and nerve protection effects, is suitable for repairing of dura mater and dura mater, and is especially suitable for minimally invasive neurosurgery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to the preparation of a low-swelling medical tissue sealant and its application in dura mater repair. Background Technology

[0002] The dura mater is the outermost protective membrane of the brain and spinal cord. Its main functions are to maintain the integrity of cerebrospinal fluid (CSF) circulation, prevent external invasion, and support intracranial and spinal canal neural structures. Dural injury is a common complication during neurosurgical and spinal surgery, with a postoperative CSF leakage rate as high as 30%. CSF leakage can lead to serious consequences such as changes in intracranial pressure, meningitis, hydrocephalus, and neurological dysfunction. Therefore, developing safe and effective dural repair materials is of significant clinical importance.

[0003] Traditional suture repair, while achieving tissue apposition, often carries the risk of needle leakage and is difficult to perform in minimally invasive or narrow surgical fields. Therefore, various commercially available sealing materials have been applied to dural repair. However, current commercially available sealing materials have significant limitations. For example, while polyethylene glycol (PEG)-based hydrogels (such as DuraSeal) offer good sealing, their high swelling rate (>60%) can easily compress adjacent nerve tissue; fibrin glue (such as Tisseel) has limited adhesive strength and is unable to withstand fluctuations in cerebrospinal fluid pressure; and bio-glue contains glutaraldehyde, posing a potential neurotoxicity risk. Autologous tissue transplantation requires additional material harvesting, increasing surgical trauma, while allogeneic or xenogeneic materials may trigger immune responses. Furthermore, hemostatic sponges rely on high swelling for sealing; although they can rapidly absorb fluid, they can cause increased intracranial or spinal canal pressure, making them unsuitable for dural repair.

[0004] In recent years, polyethylene glycol hydrogels have attracted attention due to their certain tissue adhesion properties. However, these materials still have problems such as insufficient bioactivity, low mechanical strength, and mismatch between swelling rate and degradation rate, making it difficult to meet the long-term barrier function requirements of dura mater repair.

[0005] Overall, existing dura mater repair materials still face the following major problems: excessive swelling rate, which can easily cause nerve tissue compression; insufficient mechanical strength, making it difficult to resist cerebrospinal fluid fluctuation pressure; mismatch between degradation cycle and tissue repair process, leading to sealing failure or limited tissue regeneration; lack of antibacterial properties, resulting in a high risk of postoperative infection; and difficulty in controlling gelation rate, affecting intraoperative operability and sealing effect.

[0006] Therefore, there is an urgent need to develop a novel hydrogel sealing system that combines controllable gelation rate, suitable mechanical properties, low swelling rate, matching degradation cycle, and good biocompatibility. Summary of the Invention

[0007] This invention aims to solve at least one of the technical problems existing in the prior art. The polyethylene glycol-aminated gelatin two-component system proposed in this invention, or the selective introduction of amino-rich polymers as a third component, can rapidly form gels in situ under physiological conditions, possessing high-strength sealing properties, controllable degradation characteristics, and excellent biocompatibility, providing a new technical solution for the repair of the dura mater (including the meninges and spinal dura mater).

[0008] The first aspect of the present invention is to provide a sealant.

[0009] The second objective of this invention is to provide a method for preparing the sealant of the first aspect of this invention.

[0010] The third aspect of this invention aims to provide the application of the sealant of the first aspect of this invention in the preparation of products.

[0011] The fourth aspect of this invention is to provide a product.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sealant, the raw materials for which the sealant is prepared include multi-arm polyethylene glycol active ester and aminated gelatin; wherein, The aminated gelatin is prepared by amylating gelatin with one or more substances selected from ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, and hyperbranched polyethyleneimine.

[0013] The multi-arm polyethylene glycol active ester has a structural formula of at least one of Formula 1, Formula 2, Formula 8 and Formula 10:

[0014]

[0015]

[0016] Wherein, n is independently selected from any positive integer from 1 to 1000; x is independently selected from any positive integer from 0 to 6; y is independently selected from any positive integer from 1 to 6; R is selected from sulfonic acid groups or hydrogen atoms; m is the number of arms of the multi-arm polyethylene glycol active ester, and m is independently selected from any positive integer from 2 to 4.

[0017] In some embodiments of the present invention, the active ester in the multi-arm polyethylene glycol active ester is selected from at least one of succinimidyl succinate, succinimidyl valerate, succinimidyl glutarate, succinimidyl adipate, succinimidyl octanoate, succinimidyl sebacate, N-hydroxysuccinimidyl ester, succinimidyl acetate, and succinimidyl thioglutarate. In some embodiments of the present invention, the molecular weight of the multi-arm polyethylene glycol active ester is in the range of 2-100 kDa; more specifically, 4-80 kDa.

[0018] In some embodiments of the present invention, the molecular weight of the gelatin ranges from 10 to 800 kDa; more specifically, it is 20 to 80 kDa. In some embodiments of the present invention, the gelatin is primarily derived from collagen-containing connective tissues (including bones, skin, tendons, and ligaments) of animals such as cattle and pigs, as well as the skin, bones, and scales of fish. In some embodiments of the present invention, the aminated gelatin is prepared by amylating gelatin with one or more substances selected from ethylenediamine, trilysine, 1,6-hexanediamine, trilysine, and hyperbranched polyethyleneimine.

[0019] In some embodiments of the present invention, the amino acid-modified gelatin is a compound with the structural formula shown in any one of Formulas I-IV:

[0020] Wherein, m in Equation I is independently selected from any positive integer from 1 to 5, and n in Equation II is independently selected from any positive integer from 1 to 4; It represents gelatin.

[0021] In some embodiments of the present invention, the aminated gelatin is prepared by a method comprising: mixing gelatin with one or more of the following substances: ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, and hyperbranched polyethyleneimine, dissolving the mixture in a buffer solution, adjusting the pH value, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI), and reacting to obtain aminated gelatin.

[0022] In some preferred embodiments of the present invention, the buffer solution includes at least one selected from phosphate buffer, borate buffer, histidine buffer, sodium bicarbonate-sodium carbonate buffer, Tris-HCl buffer, diethanolamine buffer, 2-morpholine ethanesulfonic acid buffer, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer and 3-(N-morpholine)propanesulfonic acid buffer, preferably phosphate buffer.

[0023] In some preferred embodiments of the present invention, the molar ratio of the carboxyl group in the gelatin to the amino group in ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, or hyperbranched polyethyleneimine is 1:(0.01-100), preferably 1:(0.1-10), such as any value or a range formed by any two of 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0024] In some preferred embodiments of the present invention, the molar ratio of the carboxyl group in the gelatin to ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, or hyperbranched polyethyleneimine is 1:(0.01-100); more preferably 1:(0.1-10), such as any value or a range formed by any two of 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0025] In some preferred embodiments of the present invention, the molar ratio of carboxyl groups to EDCI in the gelatin is 1:(1-20); further, it is 1:(1-10), such as any value of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 or any range formed by both.

[0026] In some preferred embodiments of the present invention, the pH value is adjusted to 3-8; further, the pH value is adjusted to 4-7, such as any value of 4, 5, 6 or 7 or a range formed by any two of them.

[0027] In some preferred embodiments of the present invention, the reaction time is 5-72 h; more specifically, 8-20 h, such as any value or a range formed by any two of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 h.

[0028] In some preferred embodiments of the present invention, the temperature of the reaction is 10-60°C; further, it is 30-45°C, such as any value or a range formed by any two of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45°C.

[0029] In some preferred embodiments of the present invention, the preparation method further includes dialysis and drying. The dialysis and drying can be performed using techniques well known to those skilled in the art.

[0030] In some embodiments of the present invention, the raw materials for preparation further include polyamino polymers.

[0031] This invention provides a sealant with a simple preparation process, significant low swelling characteristics, and excellent biocompatibility and biodegradability. This sealant can achieve rapid in-situ gelation under physiological conditions, possessing high burst pressure, excellent tissue adhesion, controllable degradation cycle, and low swelling characteristics, thus meeting the comprehensive clinical needs for dural defect sealing and tissue regeneration. If practical application requires, a third component (polyamine polymer) can be selectively introduced to further significantly enhance its antibacterial, anti-infective efficacy, and anti-swelling stability. The sealant can be used flexibly and in various ways, depending on the actual application scenario, including but not limited to injection, coating, and bonding.

[0032] In some embodiments of the present invention, the molecular weight of the polyamino polymer ranges from 0.4 to 300 kDa.

[0033] In some embodiments of the present invention, the polyamino polymer is selected from at least one of hyperbranched polyethyleneimine, ε-polylysine, poly-L-lysine, hyperbranched polylysine, trilysine, and dendritic polylysine.

[0034] In some embodiments of the present invention, the molecular weight of the hyperbranched polyethyleneimine ranges from 0.4 to 300 kDa; more specifically, from 0.6 to 20 kDa. In some embodiments of the present invention, the molecular weight of the ε-polylysine is 0.5 to 300 kDa; more specifically, from 0.6 to 100 kDa. In some embodiments of the present invention, the molecular weight of the poly-L-lysine is 0.4 to 300 kDa; more specifically, from 0.6 to 100 kDa. In some embodiments of the present invention, the molecular weight of the hyperbranched polylysine is 0.4 to 300 kDa; more specifically, from 0.6 to 100 kDa. In some embodiments of the present invention, the molecular weight of the dendritic polylysine is 0.4 to 300 kDa; more specifically, from 0.6 to 100 kDa.

[0035] In some embodiments of the present invention, the hyperbranched polyethyleneimine is a compound with the structural formula shown in Formula 6; the trilysine is a compound with the structural formula shown in Formula 7; the ε-polylysine is a compound with the structural formula shown in Formula 8; the poly-L-lysine is a compound with the structural formula shown in Formula 9; the hyperbranched polylysine is a compound with the structural formula shown in Formula 10; and the dendritic polylysine is a compound with the structural formula shown in Formula 11. .

[0036] In some embodiments of the present invention, the mass ratio of the multi-arm polyethylene glycol active ester to the aminated gelatin is 1:(0.01-10000); further, 1:(0.1-100); even further, 1:(0.1-10), such as any value or a range formed by any two of 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0037] In some embodiments of the present invention, the mass ratio of the multi-arm polyethylene glycol active ester to the polyamino polymer is 1:(0.001-1000); further, 1:(0.01-10); even further, 1:(0.01-1), such as any value or a range formed by any two of 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1:1.

[0038] In some embodiments of the present invention, the preparation raw materials further include buffer solution.

[0039] In some embodiments of the invention, the pH value of the buffer solution is 6-10, such as any one of 6, 7, 8, 9 or 10 or a range formed by any two of them.

[0040] In some embodiments of the present invention, the buffer solution is selected from at least one of phosphate buffer, borate buffer, histidine buffer, sodium bicarbonate-sodium carbonate buffer, Tris-HCl buffer, diethanolamine buffer, 2-morpholine ethanesulfonic acid buffer, N-(2-hydroxyethyl)piperazine-N'2-ethanesulfonic acid buffer and 3-(N-morpholine)propanesulfonic acid buffer; preferably phosphate buffer.

[0041] A second aspect of the present invention provides a method for preparing the sealant of the first aspect of the present invention, comprising the following steps: mixing multi-arm polyethylene glycol active ester with a buffer solution to obtain solution A; mixing aminated gelatin with a buffer solution to obtain solution B; mixing solution A and solution B to obtain a sealant; or mixing multi-arm polyethylene glycol active ester with a buffer solution to obtain solution A; mixing aminated gelatin and a polyamine polymer with a buffer solution to obtain solution B; mixing solution A and solution B to obtain a sealant.

[0042] The sealant provided by this invention uses safe and widely available raw materials with good biocompatibility. Its preparation process is simple and can be gelled in situ by injection, making it suitable for minimally invasive surgery.

[0043] A third aspect of the invention provides the use of the sealant of the first aspect of the invention in the preparation of a product.

[0044] In some embodiments of the present invention, the product is used for wound closure, emergency hemostasis, intraoperative hemostasis, postoperative tissue closure and leakage prevention, postoperative tissue adhesion, prevention of tissue adhesion, tissue filling, tissue repair, or as a skin dressing. In some embodiments of the present invention, the tissue includes the dura mater and the spinal dura mater.

[0045] In some embodiments of the present invention, the product includes medical materials or drugs.

[0046] A fourth aspect of the present invention provides a product comprising the sealant of the first aspect of the present invention.

[0047] In some embodiments of the present invention, the product comprises medical materials or drugs. In some embodiments of the present invention, the product is used for wound closure, emergency hemostasis, intraoperative hemostasis, postoperative tissue closure and leakage prevention, postoperative tissue adhesion, prevention of tissue adhesion, tissue filling, tissue repair, or as a skin dressing. In some embodiments of the present invention, the tissue includes the dura mater (such as the meninges and spinal dura mater). In some embodiments of the present invention, the product may be in the form of a gel or powder.

[0048] The beneficial effects of this invention are: This invention provides a sealant formed by cross-linking multi-arm polyethylene glycol active ester and aminoated gelatin via amide bonds. This sealant exhibits rapid gelation, high mechanical strength, strong tissue adhesion, ultra-low swelling rate, controllable degradation, and excellent biocompatibility. It enables in-situ sealing and repair of dural injuries, prevents cerebrospinal fluid leakage, and possesses antibacterial, anti-infective, and neuroprotective properties. It is suitable for dural and spinal dura mater repair, particularly in minimally invasive neurosurgical procedures.

[0049] Specifically, the sealant provided by this invention exhibits significantly superior tissue adhesion strength compared to fibrin glue, enabling it to form a stable and tight bond with the tissue surface, effectively ensuring the reliability of sealing applications. The sealant's compressive strength exceeds 20 MPa, providing excellent mechanical stability and ensuring a reliable physical seal on the target tissue. This not only significantly improves the success rate of sealing operations but also effectively reduces the actual amount of sealant used while maintaining sealing quality, offering significant application advantages. The sealant's burst pressure test value is greater than 700 mmHg, far exceeding the normal range of arterial blood pressure (approximately 120 mmHg), fully meeting the pressure tolerance requirements for clinical dura mater / spinal cord closure scenarios. Furthermore, the sealant degrades in vitro within 3-65 days, matching the tissue repair process. The swelling rate of the sealant is consistently controlled below 10%, exhibiting excellent low-swelling characteristics. This effectively avoids problems such as loosening of the sealing interface and degradation of mechanical properties caused by excessive swelling, preventing nerve compression and providing crucial assurance for long-term sealing reliability in clinical applications. Furthermore, this sealant exhibits excellent antibacterial properties, especially the sealant incorporating polyamino polymers, which demonstrates an inhibition rate of over 95% against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Therefore, this sealant not only rapidly closes the wound and prevents cerebrospinal fluid leakage through physical sealing, but also effectively inhibits the growth and reproduction of pathogenic bacteria around the wound due to its outstanding antibacterial properties, reducing the risk of postoperative infection from the source and significantly improving the safety and overall efficacy of clinical application. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Images of the dura mater defect in SD rats before and after sealing, as shown in Example 14 of this invention.

[0051] Figure 2 The images show the healing of the dura mater defect in the SD rats of Example 14 of this invention on days 7 and 28 after sealing.

[0052] Figure 3 MRI images of the dura mater defect in SD rats of Example 14 of this invention on days 7 and 28 after sealing.

[0053] Figure 4 Histological images of the dura mater defect in SD rats of Example 14 of this invention on days 7 and 28 after sealing.

[0054] Figure 5 Surgical images of the dura mater defect in SD rats in Example 15 of this invention before and after sealing.

[0055] Figure 6 Gross image of the back of the SD rat in Example 15 of this invention on day 7 after sealing the dural defect.

[0056] Figure 7 MRI images of the dural defects of SD rats in Example 15 of this invention on days 7 and 28 after sealing.

[0057] Figure 8 Histological images of the dural defects of SD rats in Example 15 of this invention on days 7 and 28 after sealing.

[0058] Figure 9 MRI images of a beagle with a dura mater defect sealed on day 14 in Example 16 of this invention.

[0059] Figure 10 MRI images of a beagle with a dural defect in Example 17 of this invention, taken on day 14 after sealing. Detailed Implementation

[0060] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0061] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0062] All animal procedures and protocols involved in this invention have been approved by the Laboratory Animal Ethics Committee of Southern University of Science and Technology (Approval No.: SUSTech-JY202404035).

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1: Preparation of Aminated Gelatin 2.0 g of gelatin (Sigma, 60 kDa) was dissolved in 100 mL of PBS until completely dissolved. The molar amount of carboxyl groups in the gelatin was determined by potentiometric titration (0.8 mmol / g), and the total molar amount of carboxyl groups in the system was 1.6 mmol. Ethylenediamine (3.2 mmol, 214 μL) was added, and the pH was adjusted to 5 with 1 M HCl. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) (3.2 mmol, 831.3 mg) was added. The reaction was stirred continuously at 37 °C for 12 h, followed by dialyzing with pure water for 4 days, and then lyophilized for 4 days to obtain aminated gelatin.

[0065] All subsequent embodiments of this invention involve the preparation of aminated gelatin following the process route of this embodiment; regardless of the amino donor compound used (such as ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, hyperbranched polyethyleneimine, etc.), the same preparation method (including parameters such as reaction system ratio, temperature, time, and purification steps) is used.

[0066] By selecting different amino donor compounds, the chemical formulas of the prepared aminated gelatin can be as follows: Formula I, Formula II, Formula III, or Formula IV, wherein the value of m in Formula I can be 1-5; and the value of n in Formula II can be 2 or 3.

[0067]

[0068] When m = 1 in Formula I, it is ethylenediamine modified gelatin (molecular weight 70 kDa-75 kDa); when m = 2, it is 1,3-propanediamine modified gelatin (molecular weight 72 kDa-78 kDa); when m = 3, it is 1,4-butanediamine modified gelatin (molecular weight 75 kDa-80 kDa); when m = 4, it is 1,5-pentanediamine modified gelatin (molecular weight 78 kDa-85 kDa); when m = 5, it is 1,6-hexanediamine modified gelatin (molecular weight 80 kDa-85 kDa).

[0069] When n is 1 in Formula II, it is diethylenetriamine modified gelatin (molecular weight 82 kDa-92 kDa); when n is 2, it is triethylenetetraamine modified gelatin (molecular weight 85 kDa-95 kDa); when n is 3, it is tetraethylenepentamine modified gelatin (molecular weight 90 kDa-100 kDa); when n is 4, it is pentaethylenehexamine modified gelatin (molecular weight 95 kDa-105 kDa).

[0070] Formula III corresponds to trilysine-modified gelatin (molecular weight of 80 kDa-90 kDa).

[0071] Formula IV corresponds to hyperbranched polyethyleneimine modified gelatin (100 kDa-110 kDa). The It represents gelatin.

[0072] Example 2: Determination of Amino Content Weigh out 5 mg each of gelatin and aminated gelatin (prepared from the examples) from different amino donor compounds, and dissolve them separately in 10 mL of phosphate-buffered saline (PBS) at pH 7.4 to prepare 0.5 mg / mL gelatin and aminated gelatin solutions. Take 1 mL of each solution and place it in a brown vial, then add 1 mL of 4% NaHCO3 solution and 1 mL of 0.1% sodium 2,4,6-trinitrobenzenesulfonate solution. Incubate the resulting mixture at 37°C in the dark for 2 h, and then measure the absorbance of the solution at 415 nm using a microplate reader.

[0073] Plotting the standard curve: Prepare β-aminopropionic acid solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mmol / L, respectively, and plot the standard curve of β-aminopropionic acid using the same method described above.

[0074] Calculation of amino content: Substitute the measured absorbance of gelatin and aminated gelatin into the standard curve of β-aminopropionic acid, and finally calculate the amino content in the sample.

[0075] The amino content of gelatin and aminated gelatin is shown in Table 1. The results show that the amino content of aminated gelatin is significantly higher than that of unmodified gelatin. Among them, the amino content of hyperbranched polyethyleneimine modified gelatin increased the most, which was 15 times higher than that of the original gelatin.

[0076] Table 1. Results of Amino Content Determination in Gelatin and Aminated Gelatin

[0077] Example 3-12 Prepare the sealants for Examples 3-12 according to Table 2.

[0078] Table 2. Sealants for Examples 3-12

[0079] The preparation method of the above-mentioned sealant includes the following steps: dissolving the first component and the second component in a buffer solution to obtain a first component solution (solution A) and a second component solution (solution B). Mixing solution A and solution B at a volume ratio of 1:1 yields the sealant without any additional steps.

[0080] When a third component is present, the third component is premixed into solution B and then mixed with solution A.

[0081] In practical applications, solutions A and B are separately filled into the two channels of a dual-channel syringe for later use. When needed, they are injected and mixed according to the ratio to form a gel.

[0082] The structural formula of the above four-arm polyethylene glycol succinimide succinate is shown in Formula 2:

[0083] In the formula, n is 228, y is 2, R is a hydrogen atom, m is 4, the molecular weight is 10 kDa, and it was purchased from Xiamen Sinobang Biotechnology Co., Ltd., with product number 06020702509.

[0084] The structural formula of the above-mentioned two-arm polyethylene glycol succinimide succinate is shown in Formula 2, where n is 228, y is 2, R is a hydrogen atom, m is 2, and the molecular weight is 5 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06022102506.

[0085] The structural formula of the above-mentioned six-armed polyethylene glycol succinimide thioglutarate is shown in Formula 8: where n is 74, y is 3, R is sulfonate, m is 6, and the molecular weight is 10 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06020802909.

[0086]

[0087] The structural formula of the above-mentioned two-arm polyethylene glycol succinimide valerate is shown in Formula 1: where n is 227, x is 4, R is a hydrogen atom, m is 2, and the molecular weight is 10 kDa. Purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06022102709.

[0088]

[0089] The structural formula of the above-mentioned four-armed polyethylene glycol succinimide glutarate is shown in Formula 2: where n is 57, y is 3, m is 4, R is a hydrogen atom, and the molecular weight is 10 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06020702909.

[0090] The structural formula of the above-mentioned six-armed polyethylene glycol succinimide succinate is shown in Formula 8: where n is 74, y is 2, m is 6, R is a hydrogen atom, and the molecular weight is 20 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06020802512.

[0091] The structural formula of the above-mentioned eight-arm polyethylene glycol succinimide glutarate is shown in Formula 10: where n is 14, y is 3, m is 8, R is a hydrogen atom, and the molecular weight is 10 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06021802909.

[0092]

[0093] The structural formula of the above-mentioned two-arm polyethylene glycol succinimide acetate is shown in Formula 1: where n is 228, x is 1, R is a hydrogen atom, m is 2, and the molecular weight is 10 kDa. It was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number 06022102209.

[0094] The above-mentioned trilysine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS: 13184-14-0; hyperbranched polyethyleneimine (molecular weight 10 kDa) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS: 25987-06-8; ε-polylysine (molecular weight 3.5 kDa) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS: 28211-04-3; and poly-L-lysine (molecular weight 3.5 kDa) was purchased from Sigma, CAS: 25988-63-0.

[0095] Example 13 This embodiment tests the gel time, adhesion strength, compressive strength, burst pressure, in vitro degradation, swelling rate, and antibacterial properties of the sealants from Examples 3-12, as detailed below: (1) Determination of gel time of sealant In the gel time determination of Examples 3-12, the test procedure was as follows: First, a syringe was used to draw solution A, which dissolves the first component, and another syringe was used to draw solution B, which dissolves the second and third components (if a third component is present). Then, solutions A and B (volume ratio of 1:1) were simultaneously injected into a transparent sample bottle, and the mixture was thoroughly mixed by vortexing for 3 seconds using a SN-Vortex-1 (Shanghai Shangpu Instrument Equipment Co., Ltd.). After that, the sample bottle was inverted, and the time corresponding to when the liquid in the bottle completely stopped flowing was taken as the gelation time. The relevant test results of this series of experiments are shown in Table 3.

[0096] Table 3. Results of gel time determination of sealant

[0097] As can be seen from the test data in Table 3, the cross-linking reaction rate of the system increases significantly with the increase of the amino concentration of the second component; the introduction of the third component further increases the total amino content of the system, and finally shortens the gelation time from tens of seconds to only a few seconds. This result shows that the gelation time of the system is adjustable, while the amino content of the sealant in Example 12 is insufficient, so its gelation time is slow.

[0098] (2) Determination of the adhesive strength of the sealant The adhesion strength test of the sealant in Example 3-12 was conducted as follows: Components A and B were respectively filled into the two independent channels of a dual-channel syringe. The solutions (A and B in a 1:1 volume ratio) were then injected onto the surface of the pigskin. Immediately afterward, another piece of pigskin was quickly attached to the surface coated with the mixed solution. Fibrin glue (porcine fibrin adhesive, purchased from Guangzhou Beixiu Biotechnology Co., Ltd.) was used as a control group. The treatment process was as follows: according to the instructions, the two powders of the fibrin glue were dissolved in the corresponding buffer solution. The two solutions were then filled into the dual-channel syringe and injected onto the surface of the pigskin. Immediately afterward, another piece of pigskin was quickly attached to the surface coated with the mixed solution. Both the sealant and fibrin adhesive samples were subjected to a 30-second compression force using a 100 g weight at room temperature. Following this, a shear adhesion test was performed on the pigskin samples using a universal testing machine (ETM503A, Shenzhen Wanchuang Testing Equipment Co., Ltd.) at a tensile rate of 20 mm / min until the samples broke. The maximum shear adhesion strength exhibited by the sealant was recorded during the test. The relevant experimental results are detailed in Table 4.

[0099] Table 4. Results of sealant adhesion strength test

[0100] Note: In the above table, This indicates a comparison with the fibrin glue group and Example 12. P <0.05.

[0101] The test data in Table 4 show that the tissue adhesion strength of the sealants in Examples 3-11 of this invention is significantly better than that of the control group fibrin glue (measured value 5±1 kPa). Fibrin glue has weak adhesion to tissues and is easily peeled off from the tissue surface in practical applications, resulting in poor sealing performance. In contrast, the sealants prepared in Examples 3-7 of this invention exhibit a regular increase in adhesion strength with increasing crosslinking density, forming a stable and tight bond with the tissue surface, effectively ensuring the reliability of the sealing application. The sealant in Example 12 has insufficient amino content and excessive gelatin content, resulting in lower adhesion strength.

[0102] (3) Determination of the compressive strength of the sealant The compressive strength determination process of the sealant in Example 3-12 is as follows: The corresponding solutions A and B were respectively filled into the two independent channels of a dual-channel syringe. Then, the unmixed solutions A and B (volume ratio 1:1) were injected through the syringe into a custom mold with a diameter of 5 mm and a height of 3 mm. After the system had completely gelled, the molded sample was removed. Simultaneously, fibrin glue (porcine fibrin adhesive, purchased from Guangzhou Beixiu Biotechnology Co., Ltd.) was used as a control group. The treatment process was as follows: according to its instructions, the two powders of fibrin glue were dissolved in the matching buffer solution. Then, the two solutions were filled into the dual-channel syringe and injected through the syringe into a custom mold with a diameter of 5 mm and a height of 3 mm. After the system had completely gelled, the molded sample was removed. The compressive performance of the gelled sample was tested using a universal testing machine (ETM503A, Shenzhen Wansheng Testing Equipment Co., Ltd.). The compression rate was set to 5 mm / min, and continuous loading was applied until the sample deformation reached 99% or it broke. The compressive strength data exhibited by the sealant was recorded. The relevant experimental results are detailed in Table 5.

[0103] Table 5 Results of compressive strength test of sealant

[0104] Note: In the above table, This indicates a comparison with the fibrin glue group and Example 12. P <0.05.

[0105] As shown in Table 5, the sealants prepared in Examples 3-11 all exhibit compressive strengths exceeding 20 MPa. This excellent compressive strength endows the sealants with outstanding mechanical stability, enabling them to form a reliable physical seal on the target tissue. This not only significantly improves the success rate of sealing operations but also effectively reduces the actual amount of sealant used while ensuring sealing quality, demonstrating significant application advantages. Furthermore, the experimental results show that the compressive strength of the sealant gradually increases with increasing crosslinking density. The compressive strength of the sealant in Example 12 is only 2.1 MPa, mainly due to the mass concentration ratio of aminated gelatin to polyethylene glycol active ester in its formulation (0.25 g / mL and 0.15 g / mL, respectively). The excessively high gelatin content leads to an imbalance in the crosslinking density within the material, resulting in insufficient mechanical toughness and ultimately exhibiting obvious brittleness, thus significantly reducing its compressive strength.

[0106] (4) Burst pressure test of sealant First, the surface of the fresh pigskin is cleaned to remove attached impurities and dirt. Then, the treated pigskin is cut into circular sample pieces with a diameter of 6 cm. At the center of each circular pigskin sample, a circular through hole with a diameter of 3 mm is punched. To prevent the solution from seeping into the through hole and affecting the test results during subsequent injection of sealant, the circular through hole is pre-sealed with petroleum jelly.

[0107] The specific operating procedure for the burst pressure test of the sealant in Example 3-12 is as follows: First, solution A and solution B are respectively filled into the two independent channels of a dual-channel syringe. Then, the two solutions are injected simultaneously (solution A and solution B are injected at a volume ratio of 1:1) to cover the circular through-holes in the pigskin. After the gel system has completely formed, the petroleum jelly pre-filled in the through-holes of the pigskin is carefully removed with a small spoon to restore the through-holes to a hollow state. Simultaneously, fibrin glue (porcine fibrin adhesive, purchased from Guangzhou Beixiu Biotechnology Co., Ltd.) is used as a control group. The treatment process is as follows: According to its instructions, the two powders of fibrin glue are dissolved in the matching buffer solution. Then, the two solutions are filled into the dual-channel syringe. Subsequently, the gel is injected through the syringe to cover the circular through-holes in the pigskin. After the gel system has completely formed, the petroleum jelly pre-filled in the through-holes of the pigskin is carefully removed with a small spoon to restore the through-holes to a hollow state. The treated pigskin sample was then fixed on the burst pressure test device (YB80A, Suzhou Xuansheng Instrument Technology Co., Ltd.), and the pressure resistance performance was tested by continuously injecting water. The maximum reading before the pressure dropped during the test was taken as the burst pressure test result of the sealant sample. The relevant experimental results are detailed in Table 6.

[0108] Table 6. Results of the burst pressure test of the sealant

[0109] Note: In the above table, This indicates a comparison with the fibrin glue group and Example 12. P <0.05.

[0110] As shown in Table 6, the burst pressure test values ​​of the sealants prepared in Examples 3-11 all exceeded 700 mmHg. This value is not only significantly higher than the burst pressure level of the control group fibrin glue (105±9 mmHg), but also far exceeds the normal range of arterial blood pressure (approximately 120 mmHg), which can fully meet the pressure tolerance requirements of clinical dura mater / spinal dura mater closure scenarios. In addition, it can be seen that the adhesive strength and compressive strength of the examples are synergistically improved, and their burst pressure resistance increases synchronously. Since the adhesive strength and compressive strength of the sealant in Example 12 are much weaker than those of the groups in Examples 3-11, its burst pressure resistance is also lower.

[0111] This result fully confirms that the sealant of the present invention has reliable application value in stopping leaks and sealing after damage to the dura mater and spinal dura mater.

[0112] (5) In vitro degradation test of sealant The specific operating procedure for the in vitro degradation test of the sealant in Examples 3-12 is as follows: First, solution A and solution B are respectively filled into the two independent channels of a dual-channel syringe. Then, the two solutions are simultaneously injected (at a volume ratio of 1:1) into a cylindrical glass mold with a diameter of 7 mm and a height of 3 mm, allowing them to mix thoroughly and evenly within the mold. After the system forms a gel, the formed gel is removed and transferred to a sealed container containing simulated cerebrospinal fluid. The sealed container is then placed in a 37°C constant-temperature shaker, with the shaker set to an oscillation rate of 100 r / min. The degradation process of the gel sample in the buffer solution is continuously observed until the gel sample is completely invisible to the naked eye. The recorded time is the in vitro degradation time of the gel. The experimental results are shown in Table 7 below.

[0113] Table 7. Results of in vitro degradation test of sealant

[0114] As shown in Table 7, the sealants prepared in the various embodiments of the present invention exhibit different in vitro degradation times ranging from 3 to 60 days due to variations in molecular weight and molecular structure. This allows for the selection of sealant products with specific degradation cycles to meet the actual needs of different tissue repair scenarios, thereby enabling personalized and customized treatment applications and providing more flexible solutions for clinical treatment. In Example 12, insufficient cross-linking resulted in a significantly accelerated material degradation rate, greatly limiting its adaptability to practical applications.

[0115] (6) Swelling rate test of sealant The specific operating procedure for the swelling rate test of the sealant in Example 3-12 is as follows: First, solution A and solution B are respectively filled into the two independent channels of a dual-channel syringe. Then, the two solutions are simultaneously injected into a cylindrical glass mold with a diameter of 5 mm and a height of 3 mm, allowing them to mix thoroughly and evenly within the mold. After the system forms a gel, the gel is removed and transferred to a sealed container containing simulated cerebrospinal fluid (artificial cerebrospinal fluid pH=7.4, Beijing Coolplay Technology Co., Ltd.) or PBS (pH=7.4). The sealed container is then placed in a 37°C constant-temperature shaker with a shaking rate of 100 r / min. The swelling change of the gel sample in simulated cerebrospinal fluid or PBS is continuously observed until swelling equilibrium is reached. The swelling rate is calculated as follows: Swelling rate = (mass at swelling equilibrium - mass before swelling) / mass before swelling. The experimental results are shown in Table 8 below.

[0116] Table 8. Swelling rate test of sealant

[0117] Note: In the above table, This indicates a comparison with Example 12. P <0.05.

[0118] As can be clearly seen from the test data in Table 8, the swelling rate of the sealants prepared in each embodiment of the present invention can be stably controlled within 10%, exhibiting excellent low-swelling characteristics. This effectively avoids problems such as loosening of the sealing interface and degradation of mechanical properties caused by excessive swelling of the material, providing a key guarantee for long-term sealing reliability in clinical applications. The core of this performance advantage is attributed to the fact that with the increase of amino content in the sealant system, the reaction sites with the crosslinking agent increase significantly, promoting the formation of a denser three-dimensional crosslinked network structure inside the material. This dense crosslinked network, on the one hand, restricts the penetration and diffusion of water molecules through strong interactions between molecular chains, and on the other hand, significantly reduces the free extension space of the network skeleton, thereby inhibiting the swelling behavior of the material at the molecular structure level, ultimately achieving synergistic optimization of low swelling characteristics and structural stability. As an important protective tissue in the intracranial cavity, the sealing of the dura mater after damage requires not only reliable adhesion and mechanical strength of the sealant, but also to avoid the material from expanding in volume due to excessive swelling in the in vivo environment, which could compress brain tissue or cause uneven stress on the dura mater repair site, affecting the recovery of nerve function. The low swelling properties of the sealant of this invention ensure that it maintains a stable volume shape after gelling at the site of dura mater rupture. It avoids additional mechanical pressure caused by swelling and problems such as loosening of the adhesion interface and sealing failure caused by swelling, thus significantly improving the safety and effectiveness of clinical application. In Example 12, the ratio of aminated gelatin to multi-arm polyethylene glycol active ester was unbalanced, resulting in a significantly higher swelling rate of the material.

[0119] (7) Antibacterial performance test of sealant The specific procedure for testing the antibacterial properties of the sealant in Examples 3-12 is as follows: Solution A and Solution B are first filled into the two independent channels of a dual-channel syringe, and both liquids are simultaneously injected into a 96-well plate to obtain a multifunctional hydrogel (i.e., sealant). 50 mg of the multifunctional hydrogel is then immersed in 1 mL of a bacterial suspension containing *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (approximately 1 × 10⁻⁶). 7 (CFU / mL), incubated with shaking at 37°C for 12 h. OD was measured. 600 To determine the antibacterial effect of multifunctional hydrogels on bacteria. Antibacterial rate (%) = (OD) 密封剂 -OD 空白 ) / (OD 培养板 -OD 空白 The experimental results are shown in Table 9 below.

[0120] Table 9. Test results of the antibacterial properties of the sealant

[0121] Note: In the above table, This indicates a comparison with Example 12. P <0.05.

[0122] As clearly shown by the test data in Table 9, with the gradual increase of the amino content in the sealant system of this invention, the inhibition rate of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa (Examples 6-11) remained stable at over 95%, exhibiting strong and broad-spectrum antibacterial activity. This result fully confirms that amino-modified gelatin itself endows the material with basic antibacterial properties, while the precise introduction of the third component further enhances the antibacterial efficacy, enabling the sealant to form a synergistic antibacterial mechanism and significantly improving antibacterial activity and broad spectrum. From the perspective of the mechanism of action, lysine and hyperbranched polyethyleneimine are both typical cationic polymers. The cationic groups rich in their molecular structure can interact strongly with the anionic sites on the surface of bacterial cell membranes. This interaction not only disrupts the integrity of bacterial cell membranes, leading to leakage of intracellular substances, but also further interferes with bacterial metabolic processes and proliferation cycles, ultimately achieving a highly efficient antibacterial effect. In clinical settings such as sealing dura mater and spinal cord ruptures, this sealant can not only quickly close the wound and prevent cerebrospinal fluid leakage through physical sealing, but also effectively inhibit the growth and reproduction of pathogenic bacteria around the wound due to its outstanding antibacterial properties, thereby reducing the risk of postoperative infection from the source and significantly improving the safety and overall efficacy of clinical applications.

[0123] Example 14: Sealing experiment of sealant on rat dura mater Preparation of the sealant (i.e., the sealant of Example 8): Weigh 150 mg of tetra-arm polyethylene glycol succinimide glutarate (molecular weight 10 kDa), dissolve it in 1 mL of PBS at pH 7.4 to prepare solution A (volume concentration 150 mg / mL); weigh 150 mg of aminated gelatin (ethylenediamine modified gelatin), molecular weight 60 kDa, dissolve it in 1 mL of PBS at pH 7.4 to prepare solution B (volume concentration 150 mg / mL). Fill solutions A and B into the two channels of a dual-channel syringe for later use.

[0124] The experimental procedure was as follows: First, the skin of the frontal vertex of SD rats (250-300 g, male) was prepared by removing the surface hair and then disinfected using routine methods. A longitudinal incision was then made along the midline of the head, extending posteriorly to the occipital protuberance, with a total length controlled to approximately 3 cm. Next, a circular bone defect with a diameter of 5 mm was created in the area posterior to the coronal suture and to the right of the sagittal suture using an electric abrasive device. After the dura mater was fully exposed, the dura mater and arachnoid mater were dissected layer by layer using microsurgical scissors to construct a 3 mm long animal model of a dural defect. After successful model establishment, any leaked cerebrospinal fluid from the wound was gently wiped away. Then, a sealant (solution A and solution B, volume ratio 1:1) was injected into the surface of the dural defect using a dual-channel syringe to complete the sealing operation. Simultaneously, a control group and a fibrin glue group were set up. The control group used the sealant prepared according to the formulation of Example 12. Specifically, 150 mg of two-arm polyethylene glycol succinimide succinate (molecular weight 5 kDa) was weighed and dissolved in 1 mL of PBS at pH=7.4 to prepare solution A (mass-volume concentration of 150 mg / mL); 250 mg of aminated gelatin (ethylenediamine modified gelatin), molecular weight 60 kDa, was weighed and dissolved in 1 mL of PBS at pH=7.4 to prepare solution B (mass-volume concentration of 250 mg / mL). Solutions A and B were respectively filled into the two channels of a dual-channel syringe for later use.

[0125] After the model was successfully established, the leaked cerebrospinal fluid at the wound site was gently wiped clean. Then, a sealant (solution A and solution B, volume ratio 1:1) was injected into the surface of the dural defect using a dual-channel syringe to complete the sealing operation. For the fibrin glue group, after the model was successfully established, the leaked cerebrospinal fluid at the wound site was gently wiped clean. Then, fibrin glue (porcine fibrin adhesive, purchased from Guangzhou Beixiu Biotechnology Co., Ltd.) was evenly injected into the surface of the dural defect using the original syringe.

[0126] The results are as follows Figure 1 As shown, after the sealant of this invention is injected, the material spreads, wets and solidifies rapidly to form a dense sealing barrier, which adheres firmly to the dura mater and adapts to tissue mechanical deformation. During the 30-minute observation period, there were no abnormalities such as cerebrospinal fluid leakage or bleeding. In the control group, cerebrospinal fluid continued to leak out, and the fibrin glue group was also completely sealed.

[0127] like Figure 2As shown, observations at 7 days post-surgery revealed cerebrospinal fluid leakage and mild tissue edema in both the control group and the fibrin glue group, indicating poor sealing performance. In contrast, the sealant group of this invention showed good wound healing with no leakage or abnormal reactions, demonstrating reliable sealing performance. Results at 28 days post-surgery showed significant bulging at the dura mater defect site in both the control group and the fibrin glue group (soft texture, fluctuating upon pressure, indicating cerebrospinal fluid accumulation and seal failure); while the sealant group of this invention showed smooth wound healing, good integration with surrounding tissues, and no cerebrospinal fluid accumulation or leakage.

[0128] like Figure 3 As shown, MRI scans performed 7 days post-surgery revealed extensive high-density water signals in the defect areas of the control group and the fibrin glue group (indicating significant cerebrospinal fluid leakage). In the sealant group, the defect area showed characteristic high-density signals of the material, forming a clear low-signal septum between the sealant and brain tissue, confirming the formation of a complete physical barrier and blocking the leakage pathway. MRI scans performed 28 days post-surgery showed that the control group and the fibrin glue group still exhibited leakage-related water signals, indicating complete loss of sealing performance. In contrast, the sealant group of this invention maintained its characteristic material signals and intact barrier, with no cerebrospinal fluid leakage signals. Its long-term sealing stability was significantly superior to that of the fibrin glue, providing a sufficient time window for dura mater repair.

[0129] like Figure 4 As shown, histological analysis at 7 days post-surgery revealed that the control group had cerebrospinal fluid leakage channels in the defect area but no obvious granulation tissue; the fibrin glue group only had a small amount of loosely filled, disordered granulation tissue, resulting in low repair efficiency; the sealant group regenerated a large amount of dense and well-organized granulation tissue, which was continuous with the surrounding dura mater, with no interface gaps, and excellent tissue compatibility, allowing for accelerated repair using a three-dimensional scaffold. Histological analysis at 28 days post-surgery showed that the control group had no functional dura mater regeneration, the fibrin glue group had loose connective tissue bulging due to long-term leakage, resulting in poor repair; the sealant group showed complete regeneration of structurally intact, nearly normal-shaped dura mater-like tissue in the defect area, covered with mature and dense connective tissue, resulting in a stable repair interface.

[0130] Example 15: Sealing Experiment of Sealant on Rat Dural Preparation of the sealant (i.e., the sealant of Example 8): Weigh 150 mg of tetra-arm polyethylene glycol succinimide glutarate (molecular weight 10 kDa), dissolve it in 1 mL of PBS at pH 7.4 to prepare solution A (volume concentration 150 mg / mL); weigh 150 mg of aminated gelatin (ethylenediamine modified gelatin), molecular weight 60 kDa, dissolve it in 1 mL of PBS at pH 7.4 to prepare solution B (volume concentration 150 mg / mL). Fill solutions A and B into the two channels of a dual-channel syringe for later use.

[0131] The experimental procedure was as follows: After anesthetizing SD rats (250-300 g, male), the skin of the lumbar region of their back was prepared, followed by disinfection and draping of the surgical area. A longitudinal incision was made along the midline of the back at the lumbar region, sequentially cutting the skin, subcutaneous fat tissue, and lumbar muscle layer. The spinous processes and lamina structures were exposed by blunt dissection. Using single-joint osteotomes, the spinous processes and part of the lamina of the target segment were gradually removed, fully exposing the dura mater sac. Under direct vision, the regular pulsation of the dura mater sac in sync with respiratory rhythm could be observed. Subsequently, under the assistance of a surgical microscope, the dura mater was gently lifted with micro-hooks, and the dura mater and deep arachnoid membrane were cut along the longitudinal axis using micro-scissors, controlling the incision length to approximately 1 cm. Clear cerebrospinal fluid was observed to continuously flow out immediately after the incision, indicating the successful establishment of the rat dural defect leakage model. The sealant of this invention (solution A and solution B, volume ratio 1:1) was injected into the dural wound, and a control group and a fibrin glue group were also set up. The control group used the sealant prepared according to the formulation of Example 12. Specifically, 150 mg of two-arm polyethylene glycol succinimide succinate (molecular weight 5 kDa) was weighed and dissolved in 1 mL of PBS at pH 7.4 to prepare solution A (volume concentration 150 mg / mL); 250 mg of aminated gelatin (ethylenediamine modified gelatin), molecular weight 60 kDa, was weighed and dissolved in 1 mL of PBS at pH 7.4 to prepare solution B (volume concentration 250 mg / mL). Solutions A and B were respectively filled into the two channels of a dual-channel syringe for later use. After the model was successfully established, the cerebrospinal fluid leaking from the wound was gently wiped clean, and then the sealant (solution A and solution B, volume ratio 1:1) was injected into the surface of the dural defect wound through the dual-channel syringe to complete the sealing operation. After the model was successfully established, the cerebrospinal fluid leaking from the wound was gently wiped clean. Then, the fibrin glue (porcine fibrin adhesive, purchased from Guangzhou Beixiu Biotechnology Co., Ltd.) was evenly injected onto the surface of the dura mater defect using the original syringe.

[0132] The sealant of this invention rapidly gels upon injection into the dura mater wound, forming a continuous and dense sealing layer. Postoperatively, there is no cerebrospinal fluid leakage, and the rats exhibit normal motor function. Figure 5 (The fibrin glue ruptured due to the high burst pressure of cerebrospinal fluid after short-term sealing, resulting in persistent cerebrospinal fluid leakage in both the control and fibrin glue groups, and hind limb motor dysfunction in some rats.) The results confirm that the sealant of this invention is significantly superior to traditional fibrin glue in terms of effectiveness, persistence, and biocompatibility in sealing dural defects.

[0133] like Figure 6 As shown, gross observation and palpation of SD rats 7 days after surgery: the surgical area of ​​the control group and the fibrin glue group was obviously swollen, soft and fluctuating (suggesting cerebrospinal fluid leakage and accumulation); the surgical area of ​​the sealant group was flat, uniform in texture and without fluctuation, and the seal was stable, effectively blocking the leakage of cerebrospinal fluid.

[0134] like Figure 7 As shown, MRI at 7 days post-surgery revealed significant high-density water signals in the surgical area of ​​the control group and the fibrin glue group (suggesting cerebrospinal fluid leakage and accumulation); the sealant group showed low water signals in the defect area with clear boundaries from the normal dura mater, confirming that the material blocked the leakage pathway. MRI at 28 days post-surgery showed that the control group and the fibrin glue group still had scattered moderate-intensity water signals (leakage not completely terminated); the sealant group showed complete disappearance of water signals in the defect area, with the signal consistent with normal tissue, indicating that the material had degraded and induced tissue repair.

[0135] like Figure 8 As shown, histological analysis at 7 and 28 days postoperatively revealed tissue adhesions, spinal canal stenosis, and spinal cord compression and deformation in the surgical area of ​​the control group and the fibrin glue group (consistent with hindlimb motor dysfunction); the spinal cord in the sealant group remained intact without adhesions or compression, and the sealant adhered tightly to the dura mater, ensuring the integrity of the spinal canal and a stable repair environment.

[0136] Example 16 Sealing Experiment of Sealant in Beagle Dura Masques Preparation of the sealant (i.e., the sealant of Example 9): Weigh 150 mg of six-arm polyethylene glycol succinimide succinate (molecular weight 20 kDa) and dissolve it in 1 mL of PBS at pH 7.4 to prepare solution A. Weigh 150 mg of aminated gelatin (hyperbranched polyethyleneimine modified gelatin), molecular weight 60 kDa; weigh 10 mg of hyperbranched polyethyleneimine, molecular weight 10 kDa, and dissolve it in 1 mL of PBS at pH 7.4 to prepare solution B. Fill solutions A and B into the two channels of a dual-channel syringe for later use.

[0137] Healthy beagle dogs weighing 13 kg were selected as experimental animals. Preoperatively, the dogs were fasted for 12 hours and deprived of water for 8 hours. Anesthesia was induced by intravenous propofol. After the animals lost consciousness, endotracheal intubation was performed quickly, and a ventilator was connected for assisted breathing. The tidal volume was set at 12 mL / kg to ensure ventilation efficiency. During the maintenance phase of anesthesia, 1.5% isoflurane was continuously inhaled, and vital signs were monitored in real time using electrocardiogram monitoring to ensure a stable and controllable depth of anesthesia.

[0138] Using a cranial drill, a 10 mm diameter bone hole was drilled in the pre-designated non-functional area. The bone flap was carefully removed, and the dura mater was gently lifted with a meningeal hook. A 5 mm incision defect was made with microscissors. A sealant (solution A and solution B in a 1:1 volume ratio) was injected into the defect using a dual-channel syringe to fill the bone hole. Fibrin glue was used as a control group.

[0139] like Figure 9As shown, MRI imaging results revealed a large amount of white water signal in the dural fistula area of ​​the fibrin glue group. This signal is related to the local accumulation of water after cerebrospinal fluid leakage, suggesting that the fibrin glue failed to completely seal the dural defect. In contrast, the fistula area corresponding to the sealant group of this invention showed no obvious white water signal distribution, consistent with the signal characteristics of normal dural tissue. This phenomenon directly confirms that, compared to fibrin glue, this sealant can form a tight and stable interface with the dural tissue, effectively blocking the cerebrospinal fluid leakage channel and achieving a stable seal for the dural fistula. The integrity and reliability of its seal are supported by imaging evidence.

[0140] Example 17 Sealing Experiment of Sealant on Dura Mammary Gland of Beagle Dogs Preparation of the sealant (i.e., the sealant of Example 9): Weigh 150 mg of two-arm polyethylene glycol succinimide succinate (molecular weight 5 kDa) and dissolve it in 1 mL of PBS at pH 7.4 to prepare solution A. Weigh 150 mg of aminated gelatin (hyperbranched polyethyleneimine modified gelatin), molecular weight 60 kDa; weigh 10 mg of hyperbranched polyethyleneimine, molecular weight 10 kDa, and dissolve it in 1 mL of PBS at pH 7.4 to prepare solution B. Fill solutions A and B into the two channels of a dual-channel syringe for later use.

[0141] Healthy beagle dogs weighing 13 kg were selected as experimental animals. They were fasted for 12 hours and deprived of water for 8 hours before the operation. Anesthesia was induced by intravenous propofol. After the animals lost consciousness, endotracheal intubation was performed quickly, and they were connected to a ventilator for assisted breathing. The tidal volume was set at 12 mL / kg to ensure ventilation efficiency. During the anesthesia maintenance phase, 1.5% isoflurane was continuously inhaled, and vital signs were monitored in real time by electrocardiogram monitoring. After assuming a prone position and immobilization, routine disinfection and draping were performed. A midline longitudinal incision was made at the L3-L4 intervertebral space, sequentially incising the skin, subcutaneous tissue, and paraspinal muscles to expose the lamina. A 10 mm diameter laminectomy window was created using a high-speed drill. A full-thickness defect model was constructed by incising the dura mater with a microsurgical scalpel, immediately revealing a large amount of clear cerebrospinal fluid leakage. A sealant was injected into the defect using a dual-channel syringe to fill it. Fibrin glue was used as a control group.

[0142] like Figure 10As shown, MRI results revealed a large amount of characteristic white water signals at the dural fistula site in the fibrin glue group. The formation of these signals is closely related to the abnormal accumulation of local water after cerebrospinal fluid leakage. This imaging finding indirectly indicates that the fibrin glue failed to completely seal the dural defect area. In stark contrast, no obvious white water signals were observed in the corresponding area of ​​the dural fistula in the sealant group. Its imaging characteristics were highly consistent with those of normal dural tissue, indicating that it effectively blocked the leakage path of cerebrospinal fluid, thereby achieving a stable sealing effect on the dural leak.

[0143] The preceding specific examples illustrate the present invention, but their purpose is solely to aid in understanding the technical solution of the invention and not to limit the scope of protection of the invention. For those skilled in the art, several simple deductions, formal modifications, or equivalent substitutions can be made while adhering to the core concept of the invention, and all such adjustments should be included within the scope of protection claimed by the present invention.

Claims

1. A sealant, wherein the raw materials for preparing the sealant include multi-arm polyethylene glycol active ester and aminated gelatin; wherein, The aminated gelatin is prepared by amylating gelatin with one or more substances selected from ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trilysine, and hyperbranched polyethyleneimine.

2. The sealant according to claim 1, characterized in that, The multi-arm polyethylene glycol active ester has a structural formula of at least one of Formula 1, Formula 2, Formula 8 and Formula 10: Wherein, n is independently selected from any positive integer from 1 to 1000; x is independently selected from any positive integer from 0 to 6; y is independently selected from any positive integer from 1 to 6; R is selected from sulfonic acid groups or hydrogen atoms; m is the number of arms of the multi-arm polyethylene glycol active ester, and m is independently selected from any positive integer from 2 to 4; Preferably, the active ester in the multi-arm polyethylene glycol active ester is selected from at least one of succinimide succinate, succinimide valerate, succinimide glutarate, succinimide adipate, succinimide octanoate, succinimide sebacic acid ester, N-hydroxysuccinimide ester, succinimide acetate, and succinimide thioglutarate.

3. The sealant according to claim 2, characterized in that, The molecular weight range of the multi-arm polyethylene glycol active ester is 2-100 kDa; and / or, the molecular weight range of the gelatin is 10-800 kDa.

4. The sealant according to claim 3, characterized in that, The amino acid-modified gelatin is a compound with a structural formula as shown in any one of Formulas I-IV; In Equation I, m is independently selected from any positive integer from 1 to 5, and in Equation II, n is independently selected from any positive integer from 1 to 4.

5. The sealant according to any one of claims 1-4, characterized in that, The raw materials used in the preparation also include polyamino polymers; Preferably, the molecular weight of the polyamino polymer is in the range of 0.4-100 kDa; Preferably, the polyamino polymer is selected from at least one of hyperbranched polyethyleneimine, ε-polylysine, poly-L-lysine, hyperbranched polylysine, and dendritic polylysine.

6. The sealant according to claim 5, characterized in that, The mass ratio of the multi-arm polyethylene glycol active ester to the aminated gelatin is 1:(0.01-10000); and / or, the mass ratio of the multi-arm polyethylene glycol active ester to the polyamine polymer is 1:(0.001-1000).

7. The sealant according to claim 5, characterized in that, The preparation materials also include buffer solution; Preferably, the pH value of the buffer solution is 6-10; Preferably, the buffer solution is selected from at least one of phosphate buffer, borate buffer, histidine buffer, sodium bicarbonate-sodium carbonate buffer, Tris-HCl buffer, diethanolamine buffer, 2-morpholine ethanesulfonic acid buffer, N-(2-hydroxyethyl)piperazine-N'2-ethanesulfonic acid buffer and 3-(N-morpholine)propanesulfonic acid buffer.

8. A method for preparing the sealant according to claim 7, comprising the following steps: Multi-arm polyethylene glycol active ester is mixed with buffer solution to obtain solution A; aminated gelatin is mixed with buffer solution to obtain solution B; solution A and solution B are mixed to obtain the sealant. or Mixing multi-arm polyethylene glycol active ester with buffer solution yields solution A; mixing aminated gelatin and polyamine polymer with buffer solution yields solution B; mixing solutions A and B yields the sealant.

9. The use of the sealant according to any one of claims 1-7 in the preparation of the product; Preferably, the product is used for tissue repair; Preferably, the product is used for wound closure, emergency hemostasis, intraoperative hemostasis, postoperative tissue closure and leakage prevention, postoperative tissue adhesion, prevention of tissue adhesion, tissue filling, or as a skin dressing.

10. A product comprising the sealant according to any one of claims 1-7.

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