An oxazoline polymer, its preparation method and application

Abstract

This invention relates to the field of biomedical materials technology, and discloses an oxazoline polymer, its preparation method, and its applications. The oxazoline polymer provided by this invention has a main chain containing numerous single bonds, resulting in high flexibility and exhibiting good hydrophilicity and strong polarity. Compared to conventional water-soluble polymers, the oxazoline polymer of this invention possesses both hydrogen bond donor and hydrogen bond acceptor properties in its molecular structure, enabling it to form stable intermolecular interactions with various polymers containing hydroxyl or carbonyl groups in aqueous systems. When used in polymer hydrogels, it can effectively improve the mechanical properties and stability of the hydrogels and exhibits good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an oxazoline polymer and its preparation method and application. Background Technology

[0002] Lower back pain is an extremely common condition, with most cases related to intervertebral disc degeneration. The intervertebral disc consists of a tough outer annulus fibrosus and a central, water-rich nucleus pulposus (NP). Degeneration or damage to the NP leads to loss of disc height and decreased mechanical function, resulting in chronic lower back pain. Currently, treatment options for intervertebral disc degeneration are limited. Conservative treatments (such as physical therapy and analgesics) often fail to fundamentally restore disc function, while surgical interventions (such as discectomy, vertebral fusion, and artificial disc replacement) are highly invasive and risky, and their long-term efficacy may not be superior to conservative treatments. Therefore, there is an urgent need for two minimally invasive intermediate treatments between conservative therapy and major surgery, which can both alleviate pain and rebuild disc function.

[0003] To fill this gap, "intervertebral disc nucleus pulposus replacement materials" have become a research hotspot. Among them, hydrogel materials are considered ideal nucleus pulposus replacement materials due to their high water content and controllable mechanical properties. Hydrogels can be implanted into the intervertebral disc through minimally invasive injection, forming a flexible support structure similar to the natural nucleus pulposus in vivo, with advantages such as preserving the mobility of the intervertebral disc and reducing stress on adjacent segments. Over the past few decades, various nucleus pulposus replacement schemes have been proposed and entered the experimental or clinical research stage, including pre-formed prostheses, mechanoelastic elements, and in-situ cured polymer gels.

[0004] Pre-formed hydrogel prostheses are implanted directly using pre-made polyvinyl alcohol (PVA) hydrogel, but this requires cutting the annulus fibrosus to insert a large volume of hydrogel, making it highly invasive. Mechanical prostheses utilize elastomers or metal springs for support, but cannot simulate the hydrodynamic properties of the nucleus pulposus. In-situ polymerized hydrogels, on the other hand, involve injecting liquid monomers or prepolymers into the intervertebral disc, offering a more minimally invasive and better treatment option compared to the above strategies. Hydrogels that rapidly solidify in vivo to form a gel, while allowing for fine-needle injection and avoiding large incisions, present challenges such as the potential toxicity of chemical cross-linking agents, exothermic curing processes, and the possibility of leakage due to incomplete curing. For example, ReGelTec's Hydrafil gel achieves percutaneous fine-needle implantation and in-vivo coagulation support using a heat-injectable PVA-based hydrogel, but the long-term stability and biocompatibility of its polyethylene glycol (PEG) formulation pose certain risks, and its mechanical properties need improvement. Therefore, the development of a new generation of hydrogel nucleus pulposus replacement materials is urgent. It is necessary to develop a new polymer that can improve the stability, mechanical properties and biocompatibility of hydrogel materials while ensuring injectability and in vivo support. Summary of the Invention

[0005] This invention provides an oxazoline polymer, its preparation method, and its application, to solve the problems of poor stability, mechanical properties, and biocompatibility of existing polymer-based hydrogel materials.

[0006] In a first aspect, the present invention provides an oxazoline polymer having the structure shown in formula (I): Formula (I); Formula (II); Formula (III) Wherein, R1 is selected from unsubstituted or Rx-substituted C1-C22 straight-chain alkyl, unsubstituted or Rx-substituted C3-C22 branched-chain alkyl, unsubstituted or Rx-substituted C3-C22 cycloalkyl, or unsubstituted or Ry-substituted C5-C22 glycosyl residues; Rx is selected from one or more of hydroxyl or C5-C22 glycosyl residues; Ry is selected from unsubstituted or hydroxyl-substituted C1-C22 straight-chain alkyl, or unsubstituted or hydroxyl-substituted C3-C22 branched-chain alkyl; n represents the number of repeating units, selected from an integer from 1 to 150; m represents the number of repeating units, selected from an integer from 1 to 100.

[0007] In one optional embodiment, R1 is selected from unsubstituted or hydroxylated C1-C10 straight-chain alkyl, unsubstituted or hydroxylated C3-C10 branched-chain alkyl, unsubstituted or hydroxylated C3-C22 cycloalkyl, or unsubstituted C5-C12 glycosyl residues.

[0008] In one alternative embodiment, the glycosyl residue is selected from one or more of monosaccharide residues, oligosaccharide residues, and polysaccharide residues.

[0009] In one optional embodiment, R1 is selected from unsubstituted or hydroxylated methyl, unsubstituted or hydroxylated ethyl, unsubstituted or hydroxylated n-propyl, unsubstituted or hydroxylated isopropyl, unsubstituted or hydroxylated n-butyl, unsubstituted or hydroxylated isobutyl, unsubstituted or hydroxylated n-pentyl, unsubstituted or hydroxylated n-hexyl, , , One or more of them.

[0010] In one alternative implementation, R1 is selected from... , , , One or more of them.

[0011] In one alternative embodiment, the oxazoline polymer has the following structure:

[0012] Secondly, the present invention provides a method for preparing an oxazoline polymer, comprising the following steps: polymerizing a compound with the structure shown in formula (Ⅳ) with 2-ethyl-2-azolinium to obtain a polymer with the structure shown in formula (Ⅱ); hydrolyzing the polymer with the structure shown in formula (Ⅱ) to obtain a polymer with the structure shown in formula (Ⅲ); and esterifying the polymer with the structure shown in formula (Ⅲ) with R1-OH to obtain an oxazoline polymer; wherein R1 is as defined above. Formula (Ⅳ).

[0013] In an optional embodiment, the preparation method of the compound with the structure shown in formula (Ⅳ) includes the following steps: chlorinating monomethyl succinate to obtain compound a; reacting 2-chloroethylamine hydrochloride and triethylamine with compound a in a solvent to undergo an amide condensation reaction to obtain compound b; and performing an intramolecular cyclization reaction of compound b and triethylamine in a solvent to obtain the compound with the structure shown in formula (Ⅳ).

[0014] In one alternative embodiment, the R1-OH is selected from one or more of glycerol, n-pentanol, xylitol, and inositol.

[0015] Thirdly, the present invention provides the application of the above-mentioned oxazoline polymer or the oxazoline polymer prepared by the above-mentioned preparation method in the preparation of hydrogels.

[0016] Fourthly, the present invention provides a hydrogel composition comprising, by weight, the following raw materials: The mixture consists of 10-35 parts of the above-mentioned oxazoline polymer or the oxazoline polymer prepared by the above-mentioned method, 10-20 parts of polyvinyl alcohol, 0.1-1.5 parts of polyvinylpyrrolidone, and 40-75 parts of water.

[0017] In one optional embodiment, the raw materials, by weight, include the following: The mixture consists of 10-30 parts of the above-mentioned oxazoline polymer or the oxazoline polymer prepared by the above method, 10-15 parts of polyvinyl alcohol, 0.1-1 parts of polyvinylpyrrolidone, and 49-75 parts of water.

[0018] In one optional embodiment, the polyvinyl alcohol has an average molecular weight of 1 million to 1.5 million and a degree of hydrolysis ≥99%. In one alternative embodiment, the K value of the polyvinylpyrrolidone is 27-32.

[0019] In one alternative embodiment, the oxazoline polymer has an average molecular weight of 3,000-30,000.

[0020] In one alternative embodiment, the hydrogel composition further includes 4-6 parts by weight of a contrast agent.

[0021] In one optional embodiment, the contrast agent includes at least one of barium sulfate, iohexol, iopamidol, iodixanol, iopromide, gadopentetate dimeglumine, gadobutrol, and gadobutrol.

[0022] Fifthly, the present invention provides a method for preparing the above-mentioned hydrogel composition, comprising the following steps: Step S1: Mix polyvinyl alcohol, polyvinylpyrrolidone and a portion of water, and then heat the mixture to obtain solution A; Step S2: The oxazoline polymer is mixed with another portion of water and then heated a second time to obtain solution B; Step S3: Mix solution A with solution B and allow to stand to obtain a hydrogel; Alternatively, it may include the following steps: Step S11: Mix polyvinyl alcohol, polyvinylpyrrolidone and water and then heat for the first time to obtain solution A; Step S12: The oxazoline polymer is subjected to a second heating to obtain a melt; Step S13: Mix solution A with the melt and allow it to stand to obtain a hydrogel.

[0023] The solution A should be sufficiently viscous yet flowable, and free of undissolved polyvinyl alcohol particles.

[0024] In one alternative embodiment, if the oxazoline polymer is in the form of a solid powder or granules, it can be preheated in another container at a certain temperature (e.g., 85°C) to soften it or dissolve it in a small amount of water. If the oxazoline polymer has a low melting point (some low molecular weight oxazoline polymers have a melting point of about 60°C) or is in a liquid state, it can be directly heated to a molten state for later use.

[0025] In one optional implementation, the mass ratio of a portion of the water in step S1 to another portion of the water in step S2 is 1-2:1.

[0026] In an optional implementation, steps S1 and S11 further include the step of adding a contrast agent to solution A.

[0027] In one alternative implementation, in steps S1 and S11, the temperature of the first heating is 90-100°C.

[0028] In one alternative implementation, in steps S2 and S12, the temperature of the second heating is 75-85°C.

[0029] In one alternative implementation, the mixing time in steps S3 and S13 is 3-7 minutes.

[0030] In one optional implementation, in steps S3 and S13, the settling temperature is 20-30°C and the time is 1-3 hours.

[0031] In one alternative embodiment, the mixed solution obtained before hydrogel formation is poured into a mold or kept directly in a container and allowed to cool at room temperature. As the temperature drops to room temperature, physical gelation occurs: polyethylene glycol partially crystallizes, and the oxazoline polymer and polyvinylpyrrolidone are embedded in the gel network, gradually transforming the solution into a gel-like solid. If the solution volume is large, it can be placed in a 4°C refrigerator to accelerate gel formation. After cooling and standing for about 3 hours or more, once the gel strength has initially formed, the container is tilted to allow the upper layer of free water (if present) to precipitate out. The precipitated supernatant is poured out, weighed, and recorded for material balance calculation. Typically, the supernatant mainly consists of excess water and trace amounts of unbound polymer (if present). Removing this liquid helps to increase the gel solid content and strength. The resulting hydrogel contains some water; therefore, it appears homogeneous and transparent or slightly milky white due to the presence of barium sulfate.

[0032] In an optional embodiment, steps S3 and S13 further include sterilizing the hydrogel after it has been allowed to stand. The sterilization process is carried out at a temperature of 120-135°C for 20-40 minutes. This step sterilizes the gel and simultaneously remelts it to allow it to flow, thereby forming a denser structure after standing. The resulting hydrogel has better injectability and is sterile, making it suitable for medical procedures.

[0033] In one optional embodiment, the obtained hydrogel is dispensed into sterile syringes or storage containers while it still has some fluidity (or softened by slight heating), and then sealed for storage. If the gel is too hard to dispense at room temperature, it can be softened by warming it in a water bath at 70-80°C before filling. The final hydrogel should be stored in the dark, sealed, and refrigerated to ensure its physical stability and sterility. Before use, the gel should be checked to ensure it is free from contamination and has not dried out.

[0034] In a sixth aspect, the present invention provides the use of the above-described hydrogel composition or the hydrogel composition prepared by the above-described method in the preparation of tissue-engineered biomaterials and / or drug carriers.

[0035] The application in the preparation of tissue-engineered biomaterials specifically refers to the use of the hydrogel provided by this invention as a soft tissue replacement, supplement, repair, or in spinal surgery. Soft tissue applications include intervertebral disc repair (e.g., nucleus pulposus replacement, correction of the annulus fibrosus), knee meniscus repair, or any part of a joint reconstruction system; it is also considered for use in cosmetic and reconstructive surgeries (e.g., tissue augmentation) and for the use of the hydrogel alone or in part as an adhesive in adjuvant reconstructive surgeries; it also includes the use of the hydrogel for the preparation of cell (e.g., mesenchymal stem cell) scaffolds.

[0036] The application in the preparation of drug carriers, wherein the drug carriers include growth factor (e.g., bone morphogenetic protein) binding carriers, can be achieved by using the hydrogel composition of the present invention as a supportive scaffold structure. After loading a drug that promotes bone morphological changes, the drug-loaded hydrogel is directionally delivered to the diseased bone tissue using imaging technology. This not only supports the damaged bone structure but also allows the loaded drug to be continuously and stably released into the diseased microenvironment, precisely acting on the damaged bone cells or bone tissue, promoting bone cell proliferation and differentiation, and improving the pathological state of abnormal bone morphology.

[0037] The technical solution of this invention has the following advantages: 1. This invention provides an oxazoline polymer, which is a nonionic oxazoline polymer having the structure shown in formula (I), formula (II), or formula (III). This type of polymer has a large number of single bonds in its main chain, resulting in high flexibility and exhibiting good hydrophilicity and strong polarity. Compared with conventional water-soluble polymers, the oxazoline polymers with the structures shown in formula (I), formula (II), or formula (III) possess both hydrogen bond donor and hydrogen bond acceptor properties in their molecular structure. They can form stable intermolecular interactions with various polymers containing hydroxyl or carbonyl groups in aqueous systems. Using them in polymer hydrogels can effectively improve the mechanical properties and stability of the hydrogels and also exhibits good biocompatibility.

[0038] 2. The present invention provides a hydrogel composition comprising, by weight, the following raw materials: 10-20 parts of polyvinyl alcohol, 0.1-1.5 parts of polyvinylpyrrolidone, 10-35 parts of oxazoline polymer, and 40-75 parts of water; wherein the oxazoline polymer has a structure shown in formula (I), formula (II), or formula (III). The hydrogel composition obtained by using an oxazoline polymer with a specific structure in combination with polyvinyl alcohol, polyvinylpyrrolidone, and water in the above-mentioned specific ratio exhibits good gelation characteristics, controllable crystallinity, and thermal stability. It also possesses certain elasticity and tensile strength, excellent mechanical properties, good biocompatibility, and no potential cytotoxicity.

[0039] Specifically, from a microscopic perspective, the hydrogel formed by the hydrogel composition of this invention has a typical semi-interpenetrating polymer network microstructure. In the hydrogel, a three-dimensional framework constructed from polyvinyl alcohol (PVA) crystalline regions serving as physical crosslinking points constitutes the first network. Oxazoline polymer (POx) and polyvinylpyrrolidone (PVP) segments penetrate this framework. Although there are no covalent bonds between POx and the PVA framework, extensive hydrogen bonding interactions and molecular chain entanglement create multiple and robust physical associations with the PVA network. This constructs a unique ternary composite network where rigid PVA microregions provide skeletal support, while flexible inter-chain interactions provide synergistic reinforcement. This structure enables the hydrogel material to possess both high stiffness and high toughness. Furthermore, the oxazoline polymer of this invention forms hydrogen bonds with the hydroxyl groups on its molecular chains, the hydroxyl groups on the polyvinyl alcohol molecular chains, and the carbonyl groups on the polyvinylpyrrolidone molecular chains. This not only provides skeletal support but also helps improve the overall mechanical uniformity and structural stability of the material, significantly enhancing the hydrogel's mechanical strength, gelation characteristics, crystallinity controllability, and thermal stability.

[0040] Therefore, in the hydrogel composition of the present invention, oxazoline polymer (POx), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) are indispensable, and the present invention limits the oxazoline polymer to have a specific structure. The combined use of the three can significantly improve the mechanical properties and stability of the hydrogel.

[0041] 3. The hydrogel composition provided by this invention also includes 4-6 parts by weight of contrast agent, which enables precise delivery, positioning, and dynamic monitoring of the hydrogel in vivo, making it more convenient for clinical use. From a microscopic perspective, the contrast agent particles are uniformly dispersed and immobilized in the pores of the gel network, indicating that they can be effectively captured by the composite network, ensuring the imaging effect. In addition, the selected contrast agents all have good biocompatibility and metabolizability. While exerting the imaging effect, they can be gradually metabolized and excreted by the human body, without accumulating in the body or causing adverse reactions with other components of the hydrogel, thus not increasing the burden on the patient's body and further ensuring the safety of treatment.

[0042] 4. The method for preparing the above-mentioned hydrogel composition provided by the present invention includes the following steps: S1 step: mixing polyvinyl alcohol, polyvinylpyrrolidone and a portion of water under a first heating to obtain solution A; S2 step: mixing oxazoline polymer and another portion of water under a second heating to obtain solution B; S3 step: mixing solution A and solution B, and allowing them to stand to obtain a hydrogel; or, including the following steps: S11 step: mixing polyvinyl alcohol, polyvinylpyrrolidone and water under a first heating to obtain solution A; S12 step: heating the oxazoline polymer under a second heating to obtain a melt; S13 step: mixing solution A and the melt, and allowing them to stand to obtain a hydrogel.

[0043] During the preparation process, POx, completely dissolved in the aqueous phase, competes with PVA for water molecules to form hydrogen bonds. By inhibiting excessive crystallization of PVA, the final hydrogel exhibits a more uniformly dispersed crystalline region compared to existing PEG systems, thereby further improving the gel's mechanical uniformity and extensibility. Differential scanning calorimetry (DSC) verification shows that the gel prepared by this method exhibits a melting endothermic peak in the PVA crystalline region. However, the addition of POx causes changes in the temperature and enthalpy of this peak, indicating that although the POx segments undergo microscopic phase separation from the PVA matrix, this process is constrained by the PVA crystalline network, forming phase-separated micro-regions at the nanometer to micrometer scale. These micro-regions are called soft domains. After standing, these soft domains reabsorb water and integrate into the gel. This means that the introduction of POx not only avoids a significant phase separation interface but also increases crystallinity and network stability, resulting in a single continuous phase in the final hydrogel. The preparation method provided by this invention is simple, and the resulting hydrogel exhibits excellent properties.

[0044] 5. The application of the above-mentioned hydrogel composition provided by the present invention or the hydrogel composition prepared by the above-mentioned method in the preparation of tissue-engineered biomaterials and / or drug carriers, wherein the hydrogel can be safely injected into the patient through a fine needle, and is particularly suitable for repairing or supplementing the nucleus pulposus of the intervertebral disc, having the mechanical properties required to support the intervertebral disc, and not prolapsed when the person resumes physical activity. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 The graph shows the mechanical property test results of the hydrogels obtained in each embodiment and comparative example of Experimental Example 1 of this invention; Figure 2 This is a graph showing the test results of water content and swelling ratio of the hydrogels obtained in each embodiment and comparative example of Experimental Example 3 of this invention; Figure 3 This is a comparison chart of the in-situ curing time of the hydrogels obtained in each embodiment and comparative example in Experimental Example 4 of this invention; Figure 4 The graph shows the viscoelasticity test results of the hydrogels obtained in each embodiment and comparative example of Experimental Example 5 of this invention. Detailed Implementation

[0047] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0048] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] The structures of the compounds in this invention were determined by nuclear magnetic resonance (NMR). NMR chemical shifts are given in ppm. NMR measurements were performed using an Agilent DD2 400 MHz NMR spectrometer with CDCl3 as the solvent and tetramethylsilane (TMS) as the internal standard.

[0050] The reagents used in the embodiments and comparative examples of this invention are as follows: Polyvinyl alcohol (PVA): average molecular weight ~1.25 million, degree of hydrolysis 99%, purchased from Merck, product number: 8148941001, specification: 1KG.

[0051] Polyvinylpyrrolidone (PVP) K30: K value 27-32, purchased from McLean Company, item number: P823856-10kg, specification: 10kg.

[0052] Polyethylene glycol: CAS: 25322-68-3, average molecular weight 10000, purchased from Maclean Company, item number: P815614-10kg, specification: 10kg.

[0053] Example 1-1 Oxazoline polymer A: with an average molecular weight of 3260, has the following structure:

[0054] The reaction process is as follows:

[0055] Its preparation method is as follows: (1) Monomethyl succinate (Maclean, M812982-2.5kg) (10g, 76mmol) and thionyl chloride (Maclean, T819486-2.5L) (27g, 228mmol) and N,N-dimethylformamide (DMF, Maclean, N807505-500ml) (0.55g, 7.6mmol) were mixed and reacted at 80℃ for 3h to obtain compound 1a. After the reaction was completed, it was used directly in the next step without purification. (2) 2-chloroethylamine hydrochloride (Maclean, C804868-2.5kg) (8.8g, 76mmol) and triethylamine (TEA, Maclean, T818772-500ml) (15.4g, 152mmol) were dissolved in 80ml of dichloromethane, and then compound 1a was slowly added dropwise to obtain a reaction solution; the reaction solution was reacted at -5℃ for 16h to obtain compound 1b. After the reaction was completed, it was used directly in the next step without purification. (3) Compound 1b (14.7 g, 76 mmol) was dissolved in 20 ml of methanol (Chinese medicine, 100141601), and triethylamine (15.4 g, 152 mmol) was added as a base. The intramolecular cyclization reaction was carried out at 80 °C for 24 h to form crude compound 1c (2-(2-methoxycarbonylethyl)-2-oxazoline). Then, the crude compound 1c was washed with 50 mL of water, 50 mL of saturated sodium chloride solution, and 80 mL of 1.0 M hydrochloric acid solution to remove byproducts and obtain compound 1c. (4) 2-Ethyl-2-azolin (Merck, 137456-500mL) (5g, 50mmol) and compound 1c (2.8g, 17.8mmol) were dissolved in 20ml of acetonitrile solvent, and then methyl p-toluenesulfonate (Maclean, M813161-2.5kg) (9.3g, 50mmol) was added as an initiator. The reaction was carried out for 16h under anhydrous and oxygen-free conditions at 80℃, thereby initiating the polymerization reaction between 2-ethyl-2-azolin and compound 1c to generate compound 1d with an average molecular weight of 2846. After cooling the reaction solution to room temperature, compound 1d was precipitated by slurrying with 50ml of methyl tert-butyl ether. (5) Add 15 mL of 3M sodium hydroxide methanol solution to 6.0 g of compound 1d and perform alkaline hydrolysis at 25 °C for 4 h. After hydrolysis, the ester group at the end of compound 1d is converted to a carboxyl group to obtain compound 1e with a carboxyl group at the end. After the reaction is completed, concentrate the reaction solution under reduced pressure. No purification is required. Proceed directly to the next step of the reaction. (6) Under N2 conditions, 5.5 g of compound 1e was dissolved in 10 mL of anhydrous dichloromethane. Then, under light-protected conditions, N-hydroxysuccinimide (NHS, Maclean, N811124-500g) (5.7 g, 50 mmol), N,N-dicyclohexylcarbodiimide (DCC, Maclean, N806920-500g) (10.3 g, 50 mmol) and glycerol (1 g, 10 mmol) were added. The mixture was reacted at 25 °C for 12 h. The precipitate was filtered off, and the oxazoline polymer A was repeatedly precipitated in methyl tert-butyl ether.

[0056] The proton NMR spectrum of oxazoline polymer A: 1 ¹H NMR (400 MHz, CDCl3) δ5.5 (s, 2H, -OH), δ4.5 (d, 2H, -O-CH2-), δ4.1 (m, 1H, -O-CH), δ4.0 (d, 2H, -OCH2-), δ3.5 (t, 4H, -N-CH2-CH2-N), δ3.2 (t, 2H, -CH2-N), δ2.8 (t, 4H, CO-CH2-CH2-CO), δ2.5 (s, 3H, CH3-N), δ2.0 (s, 3H, CH3-CO-), δ1.8 (q, 2H, -CH2-), δ0.9 (t, 3H, -CH3). The ¹H NMR results detected characteristic peaks of the hydroxyl group of the polyol and the ester in the target product. The chemical shifts and integrals were consistent with the theoretical structure, confirming the successful preparation of the oxazoline compound A.

[0057] Examples 1-2 Oxazoline polymer B: average molecular weight 8100, with the following structure:

[0058] The reaction process is as follows:

[0059] Its preparation method is as follows: (1) 2-Ethyl-2-azoline (Merck, 137456-500mL) (5g, 50mmol) and compound 1c (1.6g, 10mmol) were dissolved in 20ml of acetonitrile solvent, and then methyl p-toluenesulfonate (Maclean, M813161-2.5kg) (9.3g, 50mmol) was added as an initiator. The reaction was carried out for 16h under anhydrous and oxygen-free conditions at 80℃, thereby initiating the homopolymerization of 2-ethyl-2-azoline and compound 1c to generate compound 2d with an average molecular weight of 7573. After cooling the reaction solution to room temperature, compound 2d was precipitated by slurrying with 50ml of methyl tert-butyl ether. (2) Add 15 mL of 3M sodium hydroxide methanol solution to 6.5 g of compound 2d and perform alkaline hydrolysis at 25 °C for 4 h. After hydrolysis, the ester group at the end of compound 2d is converted to a carboxyl group to obtain compound 2e with a carboxyl group at the end. After the reaction is completed, concentrate the reaction solution under reduced pressure. No purification is required. Proceed directly to the next step of the reaction. (3) Under N2 conditions, 6.0 g of compound 2e was dissolved in 10 mL of anhydrous dichloromethane, and then N-hydroxysuccinimide (NHS, Maclean, N811124-500g) (5.7 g, 50 mmol), N,N-dicyclohexylcarbodiimide (DCC, Maclean, N806920-500g) (10.3 g, 50 mmol) and pentanol (Maclean, P815992-500mL) (1.0 g, 10 mmol) were added. The reaction was carried out at 25 °C for 12 h. The precipitate was filtered off, and the oxazoline polymer B was repeatedly precipitated in methyl tert-butyl ether.

[0060] The proton NMR spectrum of oxazoline polymer B: 1 ¹H NMR (400 MHz, CDCl3) δ4.3 (t, 2H, -O-CH2-), δ3.5 (t, 4H, N-CH2-CH2-N), δ3.2 (t, 2H, N-CH2-CH2), δ2.9 (t, 4H, -CO-CH2-CH2-CO), δ2.5 (s, 3H, N-CH3), δ2.0 (s, 3H, CH3-CO-), δ1.8 (m, 8H, -CH2-), δ0.9 (t, 6H, -CH2-CH3). The ¹H NMR results detected characteristic peaks of the hydroxyl group of the polyol and the ester in the target product. The chemical shifts and integrals were consistent with the theoretical structure, confirming the successful preparation of the oxazoline compound B.

[0061] Examples 1-3 Oxazoline polymer C: average molecular weight 17700, with the following structure:

[0062] The reaction process is as follows:

[0063] Its preparation method is as follows: (1) 2-Ethyl-2-azolin (Merck, 137456-500mL) (6g, 60mmol) and compound 1c (3.1g, 20mmol) were dissolved in 20ml of acetonitrile solvent, and then methyl p-toluenesulfonate (Maclean, M813161-2.5kg) (11.2g, 60mmol) was added as an initiator. The reaction was carried out for 16h under anhydrous and oxygen-free conditions at 80℃, thereby initiating the polymerization reaction between 2-ethyl-2-azolin and compound 1c to generate compound 3d with an average molecular weight of 13741. After the reaction solution was cooled to room temperature, compound 3d was precipitated by slurrying with 50ml of methyl tert-butyl ether. (2) Add 15 mL of 3M sodium hydroxide methanol solution to 7.8 g of compound 3d and perform alkaline hydrolysis reaction at 25 °C for 4 h. After hydrolysis, the ester group at the end of compound 3d is converted to a carboxyl group to obtain compound 3e with a carboxyl group at the end. After the reaction is completed, concentrate the reaction solution under reduced pressure. No purification is required. Proceed directly to the next step of the reaction. (3) Under N2 conditions, 6.5 g of compound 3e was dissolved in 10 mL of anhydrous dichloromethane, and then N-hydroxysuccinimide (NHS, Maclean, N811124-500g) (6.9 g, 60 mmol), N,N-dicyclohexylcarbodiimide (DCC, Maclean, N806920-500g) (12.4 g, 60 mmol) and xylitol (Maclean, X820544-500g) (3 g, 20 mmol) were added under light-protected conditions. The reaction was carried out at 25 °C for 12 h. The precipitate was filtered off, and the oxazoline polymer C was repeatedly precipitated in methyl tert-butyl ether.

[0064] The proton NMR spectrum of oxazoline polymer C: 1¹H NMR (400 MHz, CDCl3) δ5.5 (s, 4H, -OH), δ4.7 (dd, 3H, -CH-), δ4.0 (d, 2H, O-CH2-), δ3.5 (t, 4H, N-CH2-CH2-N), δ3.2 (t, 2H, N-CH2-CH2), δ2.9 (t, 4H, -CO-CH2-CH2-CO), δ2.7 (s, 3H, -N-CH3), δ2.0 (s, 3H, CH3-CO-), δ1.8 (m, 2H, -CH2-), δ0.9 (t, 3H, -CH3). The ¹H NMR results detected characteristic peaks of the hydroxyl group of the polyol and the ester in the target product. The chemical shifts and integrals were consistent with the theoretical structure, confirming the successful preparation of the oxazoline compound C.

[0065] Examples 1-4 Oxazoline polymer D: average molecular weight 26400, with the following structure:

[0066] The reaction process is as follows:

[0067] Its preparation method is as follows: (1) 2-Ethyl-2-azolin (Merck, 137456-500mL) (6g, 60mmol) and compound 1c (4.1g, 26mmol) were dissolved in 20ml of acetonitrile solvent, and then methyl p-toluenesulfonate (Maclean, M813161-2.5kg) (11.2g, 60mmol) was added as an initiator. The reaction was carried out for 16h under anhydrous and oxygen-free conditions at 80℃, thereby initiating the polymerization reaction between 2-ethyl-2-azolin and compound 1c to generate compound 4d with an average molecular weight of 18586. After cooling the reaction solution to room temperature, compound 4d was precipitated by slurrying with 50ml of methyl tert-butyl ether. (2) Add 15 mL of 3M sodium hydroxide methanol solution to 8.3 g of compound 4d and perform alkaline hydrolysis at 25 °C for 4 h. After hydrolysis, the ester group at the end of compound 4d is converted to a carboxyl group to obtain compound 4e with a carboxyl group at the end. After the reaction is completed, concentrate the reaction solution under reduced pressure. No purification is required. Proceed directly to the next step of the reaction. (3) Under N2 conditions, 6.5 g of compound 4e was dissolved in 10 mL of anhydrous dichloromethane, and then N-hydroxysuccinimide (NHS, Maclean, N811124-500 g) (3 g, 26 mmol), N,N-dicyclohexylcarbodiimide (DCC, Maclean, N806920-500 g) (5.4 g, 26 mmol) and inositol (Maclean, I811835-25 g) (0.47 g, 26 mmol) were added. The reaction was carried out at 25 °C for 12 h. The precipitate was filtered off, and the oxazoline polymer D was repeatedly precipitated in methyl tert-butyl ether.

[0068] The proton spectrum of oxazoline polymer D: 1 ¹H NMR (400 MHz, CDCl₃) δ5.5 (s, 4H, -OH), δ5.0 (dd, 1H, -CH-O), δ3.5 (t, 4H, N-CH₂-CH₂-N), δ3.4 (dd, 4H, -CH-O-), δ3.2 (t, 2H, -CH₂-N), δ2.8 (t, 4H, -CO-CH₂-CH₂-CO-), δ2.6 (s, 3H, CH₃-N), δ2.1 (m, 1H, CH-CH₃), δ1.8 (m, 2H, -CH₂-), δ0.9 (d, 3H, -CH₃), δ0.9 (t, 3H, -CH₃). The ¹H NMR results detected characteristic peaks of the hydroxyl groups of the polyol and the ester in the target product. The chemical shifts and integrals were consistent with the theoretical structure, confirming the successful preparation of the oxazoline compound D.

[0069] Example 2-1 This embodiment provides a hydrogel composition, which, based on a total of 100g of raw materials, includes the following raw materials: 12g of polyvinyl alcohol, 0.15g of polyvinylpyrrolidone K30, 10g of oxazoline polymer A, 5g of barium sulfate, and the balance being water.

[0070] This embodiment also provides a method for preparing and applying a hydrogel composition, comprising the following steps: (1) Weigh the above weight of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) K30, mix with 43.71g of water, heat and stir at 95°C until completely dissolved, then add 5g of barium sulfate to the solution and stir thoroughly to obtain a milky white viscous liquid. (2) Mix 10g of oxazoline polymer A with 29.14g of water and dissolve in an 80℃ water bath to obtain a transparent solution; (3) Pour the transparent solution obtained in (2) into the liquid in (1) and stir for 5 min to mix evenly; let the mixture stand at 25°C for 2 h to initially form a gel structure and discard the upper clear liquid; place the obtained product in a high-pressure steam sterilizer and treat it at 121°C for 30 min, remove it and discard the clear liquid that precipitates again, cool and solidify to obtain hydrogel HG-5.

[0071] Example 2-2 This embodiment provides a hydrogel composition, its preparation method, and its application, which is basically the same as that in Example 2-1, except that the contrast agent is omitted, and the amounts of other raw materials and the structure of the oxazoline polymer are different. Specifically, based on a total of 100g of raw materials, the raw materials in this embodiment include: 15g of polyvinyl alcohol, 0.15g of polyvinylpyrrolidone K30, 10g of oxazoline polymer B, and the remainder is water. Among them, 44.41g of water is used in step (1), and 30.44g of water is used in step (2), and the hydrogel HG-6 is finally obtained.

[0072] Example 2-3 This embodiment provides a hydrogel composition, its preparation method, and its application, which is basically the same as that in Embodiment 2-1, except that the contrast agent is omitted, the amount of each raw material, and the structure of the oxazoline polymer are different. Specifically, based on a total of 100g of raw materials, the raw materials in this embodiment include: 15g of polyvinyl alcohol, 0.15g of polyvinylpyrrolidone K30, 25g of oxazoline polymer C, and the remainder is water. Among them, 35.41g of water is used in step (1), and 24.44g of water is used in step (2), and the hydrogel HG-7 is finally obtained.

[0073] Examples 2-4 This embodiment provides a hydrogel composition, its preparation method, and its application, which is basically the same as that in Embodiment 2-1, except that the amount of each raw material and the structure of the oxazoline polymer are different. Specifically, based on a total of 100g of raw materials, the raw materials in this embodiment include: 15g of polyvinyl alcohol, 0.15g of polyvinylpyrrolidone K30, 18g of oxazoline polymer D, 5g of barium sulfate, and the remainder is water. Among them, 37.11g of water is used in step (1), and 24.74g of water is used in step (2) to prepare hydrogel HG-8.

[0074] Examples 2-5 This embodiment provides a hydrogel composition, its preparation method and application, which are basically the same as those in Examples 2-4, except that the amount of oxazoline polymer D is increased to 35g, the amount of water in step (1) is adjusted to 26.91g, and the amount of water in step (2) is 17.94g, so as to obtain hydrogel HG-9.

[0075] Examples 2-6 This embodiment provides a hydrogel composition, its preparation method and application, which are basically the same as those in Examples 2-4, except that the amount of polyvinyl alcohol is increased to 20g, the amount of water in step (1) is adjusted to 34.11g, and the amount of water in step (2) is 22.74g, so as to obtain hydrogel HG-10.

[0076] Examples 2-7 This embodiment provides a hydrogel composition, its preparation method and application, which is basically the same as that in Examples 2-4, except that the amount of polyvinylpyrrolidone K30 is increased to 1.5g, the amount of water in step (1) is adjusted to 36.3g, and the amount of water in step (2) is 24.2g, so as to obtain hydrogel HG-11.

[0077] Comparative Example 1 This comparative example provides a hydrogel composition and its preparation method and application, which are basically the same as those in Example 2-1. The only difference is that in step (1), the addition of 0.15g of polyvinylpyrrolidone K30 is omitted, while the amount of the remaining raw materials remains unchanged, and hydrogel HG-1 is finally obtained.

[0078] Comparative Example 2 This comparative example provides a hydrogel composition and its preparation method and application, which is basically the same as Example 2-1, except that the addition of 12g of polyvinyl alcohol is omitted in step (1), while the amount of the remaining raw materials remains unchanged, and hydrogel HG-2 is finally obtained.

[0079] Comparative Example 3 This comparative example provides a hydrogel composition and its preparation method and application, which is basically the same as Example 2-1, except that step (2) is omitted. Step (3) is as follows: pour the solution obtained in step (1) into the remaining water and stir for 5 minutes to mix it evenly; let the mixture stand at 25°C for 2 hours to initially form a gel structure and discard the upper clear liquid; place the obtained product in a high-pressure steam sterilizer and treat it at 121°C for 30 minutes, then remove it and discard the clear liquid that precipitates again, and finally obtain hydrogel HG-3.

[0080] Comparative Example 4 This comparative example provides a hydrogel composition and its preparation method and application, which is basically the same as Example 2-1, except that 10g of oxazoline polymer A in step (2) is replaced with 17.5g of polyethylene glycol, and the amount of the remaining raw materials remains unchanged, and finally hydrogel HG-4 is obtained.

[0081] Experimental Example 1 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were subjected to mechanical modulus testing, and the specific steps are as follows: (1) Sample preparation: The hydrogels prepared in each example and comparative example were preheated to 65°C and poured into a plate mold with multiple pores (12 pores). Each pore of the plate mold is a cylindrical cavity (10 mm in diameter and 10 mm in depth). The top is equipped with a plunger that can be tightly inserted to flatten the surface and obtain a cylindrical hydrogel sample, ensuring that the upper and lower bottom surfaces are parallel and smooth. All samples were tested within 24 hours after curing to ensure the comparability of the results. The tests were conducted at 25°C. To prevent moisture evaporation, the samples were placed in phosphate buffered saline (PBS, pH=7.4) for testing.

[0082] (2) Sample installation: The Shimadzu EZX universal tensile testing machine (equipped with a force sensor of 5N to 500N, two parallel and smooth compression plates, and a strain measurement system) is used for testing. The sample is placed precisely at the center of the compression plate below.

[0083] (3) Preloading: Move the crossbeam at a slow rate (0.1 mm / min) until the sensor detects a small contact force (0.01 N) to determine the "zero point" position. This is to eliminate the gap between the sample and the pressure plate and ensure good initial contact.

[0084] (4) Compression test: Uniaxial compression was performed at a constant strain rate. Based on the hydrogel properties and standards, the strain rate was set to 1 mm / min (corresponding to a sample with an initial height of 10 mm). This rate is slow enough to be considered quasi-static, and the influence of viscoelastic effect is ignored until the sample is significantly damaged (i.e., stress drops sharply). The Young's modulus and failure strain of the sample were recorded.

[0085] (5) Experimental results: such as Figure 1 As shown, in terms of Young's modulus, the Young's modulus of the hydrogels in the examples (HG-5~HG-11) is 0.25-0.55 MPa, indicating that the hydrogels provided by the present invention can effectively support the height of the intervertebral disc. Among them, the Young's modulus of Examples 2-4 (HG-8) is the largest; while the Young's modulus of the hydrogels in Comparative Examples 1-4 (HG-1~HG-4) is reduced to 0.1-0.2 MPa due to the omission or replacement of some raw materials of the hydrogel. Regarding the failure strain, the failure strain of the hydrogels in the examples (HG-5~HG-11) was significantly higher than that of the comparative examples (HG-1~HG-4), exceeding 65%. In particular, the failure strain of example HG-8 reached as high as 85%. This demonstrates that the hydrogel provided by this invention can effectively break the structural brittleness of the original network, exhibiting excellent ductility and fracture resistance. The high failure strain means that the hydrogel will not be damaged under large deformations, possessing good toughness and will not fracture brittlely even exceeding physiological loads, thus laying a mechanical foundation for its application in the human spine.

[0086] Experimental Example 2 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were subjected to tensile strength tests, and the specific steps are as follows: (1) Sample preparation and storage: The hydrogels prepared in each example and comparative example were preheated to 65°C and then poured into a custom-made dumbbell-shaped Teflon mold to cool and solidify, thus preparing dumbbell-shaped hydrogel samples. The mold dimensions were: diameter × width × height = 30mm × 2mm × 3mm, to ensure a consistent specific surface area. Before testing, the samples should be stored in PBS buffer. When testing, the samples should be taken out and the excess moisture on the surface should be gently blotted with filter paper before testing.

[0087] (2) Sample installation: Use Shimadzu EZX universal tensile testing machine (equipped with a force sensor of 50N to 100N), pneumatic flat pressure clamp (use pneumatic clamp because the hydrogel will become thinner during the stretching process, and manual clamp is prone to loosening and slipping due to the reduction in thickness), and anti-slip pad (attach a corrugated rubber pad to the clamp surface to prevent the hydrogel sample from slipping off).

[0088] (3) Preloading: The stretching speed is set to 25 mm / min, and the initial length of the dumbbell-shaped narrow section is accurately measured and recorded as the initial gauge length (L0).

[0089] (4) Test procedure: The sample is held vertically, and an initial tensile force of 0.005 N is applied to straighten the sample. The sample is stretched continuously until it breaks, and the fracture length at the point of fracture, as well as the tensile strength (MPa) and fracture energy (MJ / m) on the instrument are recorded. 3 The elongation at break is calculated using the formula: (break length - L0) / L0 × 100%.

[0090] (5) Experimental results: As shown in Table 1, the tensile test of the dumbbell-shaped sample further confirmed the compactness of the structure. Compared with each comparative example, the hydrogel toughness of each example was significantly improved, the structure was compact and the extensibility was good. Among them, Examples 2-1 to 2-4 improved the tensile strength of each example by limiting the amount of any one of PVA, PVP and POx raw materials within the preferred range. In particular, the tensile strength of Examples 2-4 (HG-8) reached 0.65 MPa, which is an order of magnitude improvement compared to the comparative examples (such as HG-2, which has a tensile strength of 0.05 MPa). At the same time, HG-8 also exhibited an elongation at break of up to 550%, demonstrating the best combination of the highest strength and excellent toughness. This is due to the synergistic effect of physical hydrogen bonding (provided by PVA) and chemical crosslinking (provided by POx). In contrast, the hydrogel of Comparative Example 1 has low strength and poor toughness, the hydrogel of Comparative Example 2 is the most prone to breakage, the hydrogel of Comparative Example 3 has weak physical entanglement, and Comparative Example 4 has slightly better performance than the other comparative examples, but is still significantly worse than the other examples.

[0091] Table 1. Tensile strength test results

[0092] Experimental Example 3 The equilibrium water content and swelling ratio of the hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested respectively. The specific steps are as follows: (1) Sample preparation and storage: The hydrogels prepared in each example and comparative example were used to prepare cylindrical hydrogel samples according to the method in Experimental Example 1, using molds of the corresponding size. The dimensions were: diameter × height = 10 mm × 5 mm, to ensure a consistent specific surface area. After gently absorbing the free water on the surface of the hydrogel sample with filter paper, it was weighed immediately, and this weight was recorded as the initial wet weight. Phosphate buffered saline (PBS, pH=7.4) was used as the soaking medium to simulate the body fluid environment. All soaking experiments were carried out in a constant temperature incubator at a temperature of 37°C to simulate the internal environment of the human body.

[0093] (2) Swelling test: Immerse the sample in a sufficient amount (usually more than 50 times the sample volume) of preheated (37°C) soaking medium. Remove the sample at set time points (0.5, 1, 2, 4, 8, 24, 48h), quickly blot the surface liquid with filter paper, weigh immediately, and record the wet weight at time t (W). t After weighing, quickly return the sample to the original medium. When W is measured twice consecutively... t When the change is less than 2%, swelling equilibrium is considered to have been reached, and the final mass at this point is recorded as the equilibrium wet weight (W). 湿 After reaching equilibrium, the sample was placed in a vacuum drying oven and dried at 60°C until constant weight (weighed every 2 hours, with a weight change of less than 0.5%). The final dry weight (W) was recorded.干 Swelling ratio (Q): Usually expressed as mass swelling ratio: Q = W t / W 干 .

[0094] (3) Equilibrium moisture content test: This test can be performed simultaneously with the swelling test. Another set of parallel hydrogel samples are soaked under the same conditions until swelling equilibrium is confirmed. The final mass at this point is recorded as the equilibrium wet weight (W). 湿 Subsequently, the sample was vacuum dried to constant weight to obtain the dry weight (W). 干 The formula for calculating equilibrium water content (EWC) is: EWC(%) = [(W... 湿 -W 干 ) / W 湿 ]×100%.

[0095] (4) Experimental results: such as Figure 2 As shown, the HG-5 to HG-11 hydrogels have a water content of 55–59% under equilibrium conditions, which is comparable to the initial water content of the formulation. Unlike dry hydrogels, the gels do not absorb excessive water and swell; their swelling ratio Q≈1.0–1.05 (i.e., the maximum swelling does not exceed 5% of the initial volume). Simultaneously, due to the partial crystallization of PVA preventing network dissolution, the mass loss of the gel in water is minimal (<3%), mainly likely due to the dissolution of a very small amount of unbound POx or PVP. These indicators suggest that the gels are dimensionally stable after implantation, with no risk of swelling or compression.

[0096] Experiment Example 4 The in-situ curing time of the hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 was tested respectively. The specific steps are as follows: (1) Testing equipment: A TA Instruments rheometer equipped with a Peltier temperature control system was used, employing a flat plate geometry with a diameter of 20 mm. High-precision thermocouples were also used to monitor the internal or surface temperature of the sample.

[0097] (2) Sample loading: 300 μL of homogeneous hydrogel solution preheated to 65 °C was rapidly and accurately loaded onto the bottom plate of the rheometer.

[0098] (3) Temperature program: Starting from 65°C, rapidly cool down to 37°C at a rate of 0.5°C / second and maintain for at least 30 minutes. To prevent moisture evaporation during the test, install a sealing cover around the sample and introduce water-saturated nitrogen into the cover.

[0099] (4) Parameter settings: strain is set to 1%; angular frequency: set to 1 rad / s (simulating slow stress under physiological conditions); according to the above sample loading amount, the gap between the parallel plates is set to 1000 μm.

[0100] (5) Test method: Start timing the instant the temperature reaches 37°C, continuously monitor and record the changes in storage modulus (G') and loss modulus (G'') over time for at least 30 minutes. Gel point determination: The point at which the storage modulus (G') and loss modulus (G'') intersect during the test, and G' begins to be consistently greater than G'' (G'=G''). This point marks the transition of the material from being primarily a viscous liquid to primarily an elastic solid; record the corresponding time.

[0101] (6) Experimental results: such as Figure 3 As shown, the curing time of the comparative hydrogels (HG-1~HG-4) was 28-38 minutes; compared with each comparative example, the curing time of the hydrogels (HG-5~HG-11) in each embodiment was significantly shortened (7-13 minutes), and all of them could be completely cured within 15 minutes; indicating that the three components of the present invention have a synergistic effect, enabling the hydrogel to have rapid in-situ curing performance.

[0102] Experimental Example 5 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their viscoelasticity. The specific steps are as follows: (1) The sample preparation was the same as in Experiment 1. The testing equipment used was the high-performance Shimadzu EZX universal tensile testing machine, which is equipped with dynamic testing and cyclic compression testing functions.

[0103] (2) Sample loading: The sample is securely mounted on the test platform and a small static pre-compression strain of 5% is applied to ensure that the sample is in full contact with the indenter and to simulate preloading under in-situ conditions.

[0104] (3) Dynamic strain amplitude: After the strain scan is performed to determine the linear viscoelastic region, a fixed strain amplitude of 0.5% within the linear region is selected.

[0105] (4) Parameter settings: Cycle frequency: 1Hz; Strain range: Set a strain range between physiological load and ultimate load, from 5% to 15% of compressive strain, i.e., average strain 10% and amplitude 5%; Number of cycles: 1000 times. Start the test and run the set number of cycles continuously. The machine will continuously record load-displacement data.

[0106] (5) Performance evaluation: After completing 1000 cycles, a dynamic frequency scan test is immediately performed. The energy storage modulus (E') and loss modulus (E'') at 1 Hz are read from the frequency scan curve, and the loss factor (tanδ) at 1 Hz is calculated: tanδ=E'' / E'.

[0107] (6) Experimental results: such as Figure 4 As shown, compared to the comparative examples, the E' storage modulus of the hydrogels (HG-5~HG-11) in each embodiment is improved, with HG-7~HG-9 reaching 0.45-0.55 MPa. The storage modulus of the comparative hydrogels (HG-1~HG-4) is lower, indicating that the hydrogel provided by the present invention has better elastic support capability. The tanδ loss factor of the hydrogels (HG-5~HG-11) in the embodiments is in the range of 0.08-0.11, indicating that elasticity is dominant, energy loss is small, and it is not easy to generate heat or accumulate deformation under cyclic loading. Among them, the hydrogels (HG-7~HG-9) in the embodiments balance higher modulus and lower loss, exhibiting optimal viscoelasticity.

[0108] Experimental Example 6 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their in vitro cytotoxicity. The specific steps are as follows: (1) Sample preparation: Take a specific polytetrafluoroethylene (PTFE) mold (10 mm inner diameter, 2 mm depth), and quickly inject the hydrogels of each embodiment and comparative example preheated to 65 °C into the mold hole; then, place the mold at 4 °C for 60 min to allow it to completely solidify at low temperature, obtaining a disc-shaped hydrogel sample; then remove the disc-shaped hydrogel sample from the mold, sterilize it and use it for later use. Preparation of extract for experimental group: The disc-shaped hydrogel samples of each embodiment and comparative example are prepared according to the ratio of the surface area of ​​the hydrogel sample to the extraction medium of 3 cm². 2 1 mL of the sample was immersed in the extraction medium and soaked at 37°C for 24 hours. The hydrogel sample was then discarded, and the remaining liquid was transferred to a sterile centrifuge tube. The tube was centrifuged at 3500 rpm for 5 minutes to remove any trace suspended matter. The supernatant was then filtered through a sterile 0.22 μm polyethersulfone (PES) needle filter for sterilization. The resulting filtrate was the experimental group extract. The extraction medium was α-MEM complete medium containing 10% fetal bovine serum. Negative control group extract: High-density polyethylene (Sigma-Aldrich, GF11115751) was used as the negative control. The extraction medium was the same as above, with a ratio of high-density polyethylene to extraction medium of 3 cm⁻¹. 2 After soaking 1 mL of high-density polyethylene sample at 37°C for 24 hours, the sample was discarded, and the extract was obtained by centrifugation and filtration in the same manner as the experimental group.

[0109] (2) In vitro cytotoxicity test: The test was conducted in accordance with the international standard ISO10993-5, using mouse fibroblast L929 cells, and the cells were incubated at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded per well in 96-well plates, with 100 μL of α-MEM complete medium (Gibco (Thermo Fisher Scientific), 12571063) containing 10% fetal bovine serum added to each well. After 24 h of culture, the cells were randomly divided into the following four groups and administered the following drugs: Negative control group: 100 μL / well of high-density polyethylene extract; Positive control group: 100 μL / well of complete medium containing 0.5% (v / v) phenol (Sigma-Aldrich, P1037); Blank group: 100 μL / well of complete medium; Experimental group: 100 μL / well of serially diluted hydrogel extracts from each example and comparative example (concentrations of 100%, 50%, and 25%, respectively, with the 50% and 25% concentrations diluted using complete medium). Test method: MTT assay: After culturing for 24 h following drug administration, 20 μL of MTT reagent (Sigma-Aldrich, M2128, 5 mg / mL) was added to each group. The absorbance (OD value) of live cells at 570 nm wavelength was measured using a microplate reader to convert MTT to formazan. The relative cell proliferation rate (RGR) was calculated as follows: RGR (%) = (OD value / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / (MTT / MTT) ... 实验组 -OD 空白组 ) / (OD 阴性对照组 -OD 空白组 ) ×100%, where OD 实验组 The absorbance value measured for the experimental group is OD. 空白组 The absorbance values ​​measured for the blank group are OD values. 阴性对照组 The absorbance values ​​were measured for the negative control group. Cell morphology observation: Cell morphology, adhesion, and density were directly observed under a microscope after 24 hours of culture.

[0110] (3) Evaluation criteria: Grade 0 (no cytotoxicity): RGR ≥ 100%, cell morphology is normal. Grade 1 (mild cytotoxicity): 100% > RGR ≥ 80%, most cells adhere well and the morphology is basically normal, which is within the acceptable range, but a small number of cells are rounded, the number of granules in the cytoplasm is increased, the refractive index is slightly decreased, and a very small number of floating cells are occasionally seen.

[0111] (4) Experimental Results: As shown in Table 2, after culturing the cells of all hydrogels (HG-5~HG-11) in the extraction solution for 24 hours, the cells showed normal morphology and good adhesion, with RGR ≥100%, reaching the highest standard of Grade 0 (no cytotoxicity). They even showed a slight cell proliferation-promoting effect, indicating that the hydrogels provided by this invention fully meet the implantation requirements. The cytotoxicity grade of the comparative hydrogels (HG-1~HG-4) was Grade 1, and the cell morphology was basically normal, but a small number of cells were rounded and the number of intracytoplasmic granules increased. Although this was within the acceptable range, the performance was worse than that of the examples. This may be related to the fact that its imperfect network structure led to the dissolution of more uncrosslinked molecules.

[0112] Table 2 Results of in vitro cytotoxicity tests

[0113] Experimental Example 7 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their blood compatibility. The specific steps are as follows: (1) Sample preparation: The preparation method is basically the same as that of the extract in Experiment 6, except that the extraction medium in this experiment is physiological saline (i.e., sodium chloride solution with a concentration of 0.9%).

[0114] (2) In vitro hemolysis test: The test was conducted in accordance with the international standard ISO10993-4. Blood sample: freshly collected rabbit anticoagulated blood (using sodium citrate as anticoagulation). Experimental groups: negative control group, positive control group and experimental group 1-11.

[0115] For experimental groups 1-11, anticoagulated whole blood and physiological saline were first mixed at a volume ratio of 1:9, and then diluted to obtain a 10% (v / v) red blood cell suspension. The diluted red blood cell suspension was then incubated with the hydrogel extract of each example and comparative example at a volume ratio of 1:4 at 37°C for 60 min. After centrifugation, the supernatant was collected, and the absorbance (OD value) of hemoglobin was measured at a wavelength of 540 nm. The negative control group used an equal volume of physiological saline instead of the hydrogel extract, and the positive control group used an equal volume of deionized water instead of the hydrogel extract. Hemolysis rate (%) = [(OD value)] 实验 -OD 阴性 ) / (OD 阳性 -OD 阴性 )]×100%, where OD 实验 The absorbance and OD values ​​measured for the experimental group are as follows: 阴性 The absorbance and OD values ​​were measured for the negative control group. 阳性 The absorbance was measured in the positive control group.

[0116] (3) Evaluation criteria: The hemolysis rate should be <5%.

[0117] (4) Experimental results: As shown in Table 3, the hemolysis rate of all hydrogels (HG-5~HG-11) in the embodiments is far lower than the international standard of 5%. Among them, HG-8 has the lowest hemolysis rate, which is only 0.7%. This data strongly proves that the hydrogel material provided by the present invention has no hemolytic effect and has high safety when in contact with blood.

[0118] Table 3 Results of in vitro hemolysis test

[0119] Experimental Example 8 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their rheology and extrusion thrust, respectively. The specific steps are as follows: (1) Testing Equipment and Materials: Thermal stability testing was performed using a rotational rheometer equipped with a parallel plate clamp (25mm diameter). Injection simulation was performed using a Shimadzu EZX universal tensile testing machine: equipped with a load cell of at least 500N and an accuracy better than 0.1N. Injection System Components (Critical): Syringe: Standard 5mL medical syringe. Tubing: 40cm long polytetrafluoroethylene (PTFE) tubing with an inner diameter of 1.0mm, used to simulate the long passage from the syringe to the surgical site. Injection Needle: 17G (standard specification, 1.07mm inner diameter), connected to the end of the PTFE tubing. Heating Jacket: used to wrap the syringe and PTFE tubing and accurately maintain the temperature at (65±1)℃.

[0120] (2) Test method: The hydrogels prepared in each example and comparative example were preheated at 65°C. Using a syringe preheated at 65°C, 2.0 mL of hydrogel sample was accurately drawn. The PTFE tubing and 17G needle were filled with preheated purified water to remove air from the system. The syringe, PTFE tubing and needle were securely connected, and the entire flow system (from the syringe barrel to the needle tip) was kept in a 65°C heating jacket and kept at a constant temperature for at least 5 minutes to allow the system temperature to be completely uniform. The assembled injection system was vertically fixed on the base of the universal tensile test, and the universal tensile test push rod was aligned with and in contact with the piston of the syringe. The crossbeam of the universal tensile test was set to move downward at a constant speed of 50 mm / min to push the syringe piston. The test was started, and the data of thrust, maximum extrusion thrust and piston displacement (mm) were continuously recorded until 2 mL of gel was completely extruded. The average value F was calculated by taking the average value of the stable segment in the thrust-displacement curve. avg According to the principles of fluid mechanics, apparent viscosity (η) app It is proportional to the average thrust, and the calculation formula is: η app = K×F avg , where K is the instrument constant. The simulated injection pressure is calculated based on the maximum thrust (N) recorded during the test and the syringe piston area.

[0121] The sample, preheated to 65℃, was loaded onto the bottom plate of the rheometer, with a test gap set to 1.0 mm (to ensure sufficient sample filling without excessive overflow under the 25mm clamp). After starting the test, the shear frequency was set to 1.0 Hz, and the strain was controlled within the 1% linear viscoelastic region, with a time scan lasting 120 minutes. During the experiment, the rheometer continuously recorded the storage modulus G' and loss modulus G''. The thermal stability time was defined as the time during which G' remained within ±10% of the initial value G'0 (i.e., the average value of the first 1-5 minutes after sample loading).

[0122] If the temperature remains within this range for 120 minutes and there is no crossover between G' and G'', and the modulus remains stable (i.e., the slopes of both G' and G'' change over time are close to zero), then the thermal stability time is recorded as >120 min, indicating that the system has not undergone significant premature crosslinking or thermal degradation at 65℃; if G'>1.1 G'0, it indicates that the system has undergone crosslinking or solvent evaporation at 65℃; if G'<0.9 G'0, it indicates that the system has undergone degradation.

[0123] Comprehensive injectability evaluation criteria: A simulated injection pressure < 80 kPa and a thermal stability time > 120 min is rated as excellent; a simulated injection pressure ≤ 100 kPa and a thermal stability time > 120 min is rated as moderate; and a simulated injection pressure > 100 kPa or a thermal stability time < 120 min is rated as poor. 100 kPa is the dividing line for smooth clinical handheld injection; a simulated injection pressure < 100 kPa indicates high smoothness for clinical handheld injection. The length of the thermal stability time determines whether the material is suitable for actual clinical use; a thermal stability time > 120 min indicates suitability for actual clinical use. Comprehensive injectability evaluation requires consideration of both physical maneuverability and thermal stability.

[0124] (3) Experimental Results: As shown in Table 4, the hydrogels of the examples (HG-5~HG-11) have moderate apparent viscosity and require lower extrusion force, resulting in smooth injection and better injectability, meeting clinical operability requirements. In particular, although HG-8 has a higher apparent viscosity, its injection pressure is still below 100 kPa, demonstrating its excellent injectability and indicating that HG-8 maintains fully feasible injectability while possessing optimal physicochemical properties. Although HG-9 to HG-11 have acceptable performance, their maximum extrusion force and simulated injection pressure are close to the comfortable range of conventional handheld injection, and their injectability score is moderate. HG-2 has the worst tensile strength and thermal stability, indicating that the use of both PVA and PVP as raw materials to prepare hydrogels leads to insufficient performance. All hydrogels of the examples exhibit thermal stability well beyond 2 hours, fully meeting the requirements of clinical operation. In summary, the hydrogel provided by the present invention maintains good injectability while its comprehensive performance (such as mechanical strength and thermal stability) is significantly better than that of traditional PEG / PVA / PVP hydrogel systems (such as the hydrogel of Comparative Example 4) or PVA / PVP hydrogels (such as the hydrogel of Comparative Example 3).

[0125] Table 4. Rheological and extrusion thrust test results

[0126] Experimental Example 9 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their gelation properties. The specific steps are as follows: (1) Test equipment and conditions: Discovery series rheometer, equipped with a Peltier temperature control system with a temperature control accuracy better than ±0.1℃. Fixture system: parallel plate fixture, diameter 25mm. Test gap: set to 1.0mm. Dynamic oscillation parameters: strain: 2% (this strain value must be within the linear viscoelastic region and determined by previous strain scans). Frequency: 1Hz.

[0127] (2) Sample loading: Take 0.55 mL of the hydrogel prepared in each example and comparative example, preheat it to 65°C to form a homogeneous hydrogel solution, and quickly and accurately load it onto the bottom plate of the rheometer. Immediately lower the upper plate to the set gap of 1.0 mm, and quickly trim off the excess sample at the edges with a preheated scraper to prevent edge effects. Maintain the temperature at 65°C for 2 minutes to allow the sample temperature to become completely uniform and to eliminate historical stress and air bubbles generated during loading.

[0128] (3) Temperature equilibrium: Set the temperature scanning program: start from 65℃ and scan to 20℃ at a constant cooling rate of 2℃ / min. This cooling rate needs to be slow enough to approach thermal equilibrium, but not too slow to avoid excessive experimental time.

[0129] (4) Judgment criteria: When the energy storage modulus (G') exceeds the loss modulus (G''), it is considered that gelation is complete. The temperature at this time is recorded as the gelation temperature, and the energy storage modulus at this time is the equilibrium energy storage modulus.

[0130] (5) Experimental results: As shown in Table 5, the gelation temperature of the comparative hydrogels (HG-1~HG-4) was relatively high and the equilibrium storage modulus was low; while the gelation temperature of the example hydrogels (HG-5~HG-11) was even lower and the storage modulus was higher. The equilibrium storage modulus of comparative examples 1 and 2 was low and the network strength was weak, forming only a weak gel network. The raw materials of comparative example 3 (HG-3) were PVA and PVP. Its gelation occurred at a higher temperature and mainly relied on physical entanglement and hydrogen bonding. Although its modulus was higher than that of HG-2, its binary system lacked chemical cross-linking due to the lack of PVA, resulting in a decrease in the cross-linking density of the hydrogel and poor performance. Comparative example 4 was better than the other comparative examples, but it was still worse than the examples.

[0131] The equilibrium storage modulus of all the hydrogels in the examples (HG-5~HG-11) was significantly higher than that of the comparative hydrogels (HG-1~HG-4). This directly demonstrates a significant synergistic effect among POx, PVA, and PVP, enabling the formation of a denser and more stable three-dimensional network structure. POx provides the main thermally reversible chemical crosslinking, the long chains of PVA strengthen and toughen the network through strong hydrogen bonding, and trace amounts of PVP improve the compatibility and hydrophilicity of the components. POx plays a dominant role in the hydrogel system. When the PVA content is fixed at 15%, compared with HG-8, the increase in POx content in HG-6 significantly improves the equilibrium storage modulus. This is because POx is the main builder of the cross-linking network, and its content directly increases the cross-linking density, thereby greatly improving the mechanical strength of the gel. As the POx content increases, the performance of the hydrogel also increases. However, the POx content is not positively correlated with G'. When the POx content is further increased (i.e., HG-9), the equilibrium storage modulus decreases. This indicates that a high POx content may make the hydrogel cross-linked tightly and the network brittle. Further increasing PVA (HG-10) or PVP (HG-11) may also slightly affect the network balance of the hydrogel, resulting in a slight decrease in performance, but it is still better than the comparative example.

[0132] Table 5. Results of gelation property tests

[0133] Experimental Example 10 The crystallinity of the hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 was tested, and the specific steps are as follows: (1) Test instruments and conditions: A TA DSC2500 differential scanning calorimeter (DSC) with a liquid nitrogen cooling system was used to ensure precise temperature control. Sample cell: Standard Tzero aluminum sealed sample cell.

[0134] (2) Sample preparation: Take the fully cured hydrogel of each example and comparative example, cut it into tiny thin slices with a blade, and accurately weigh 8.0±0.5 mg of the dry gel and place it in the sample cell. Reference: Use an empty, sealed aluminum sample cell as a reference.

[0135] (3) Test Procedure: The entire test was conducted under a nitrogen atmosphere to prevent oxidation. First Stage: Initial heating to eliminate any initial thermal history generated during sample preparation and storage, and to simulate an autoclaving process. Specifically, heating from room temperature to 121°C at a rate of 5.5°C / min, and holding at 121°C for 15 minutes to ensure complete melting of the PVA crystals. Second Stage: Programmed cooling to allow the polymer chains to recrystallize orderly from the melt at a controlled cooling rate, forming a comparable crystal structure. Specifically, cooling from 121°C to 40°C at a rate of -5.5°C / min, and holding at 40°C for 15 minutes to ensure complete crystallization and equalize the instrument temperature. Third Stage: Secondary heating to measure the melting behavior of the sample after standard thermal history treatment, obtaining repeatable and comparable crystallinity data. Specifically, heating from 40°C to 250°C at a rate of 5.5°C / min, recording key endothermic melting peaks during this stage.

[0136] (4) Experimental results: The results are shown in Table 6. The crystallization temperature (Tc) of the hydrogels in the examples (HG-5~HG-11) is between 90-95℃ and the melting enthalpy (ΔHm) is between 55-95J / g, while the crystallization temperature (Tc) of the comparative hydrogels (HG-1~HG-4) is between 82-85℃ and the melting enthalpy (ΔHm) is between 45-52J / g. Higher crystallization temperature and greater endothermic effect are signs of a more stable gel. When the gel composition is heated above its melting temperature, a gel-sol transition occurs, resulting in disintegration and destruction of the bonds in the crystalline network structure. The hydrogel system provided by this invention has better performance because the addition of POx to PVA forms a crystalline network structure that does not tend to disintegrate.

[0137] The comparative hydrogels (HG-1~HG-4) showed lower crystallization temperatures and smaller enthalpies of melting. The example hydrogels (HG-5~HG-8) exhibited increased crystallization temperatures and significantly increased enthalpies of melting, indicating that the addition of POx increased crystallinity and network stability; HG-8 showed the highest crystallization temperature and enthalpy of melting. Increasing the amounts of POx, PVA, and PVP (HG-9~HG-11) respectively led to a decrease or reduction in enthalpy of melting and crystallization temperature, suggesting possible phase separation or plasticizing effects in the system, but the performance was still better than that of the comparative hydrogels. The melting temperature (Tm) of the example hydrogels was also slightly higher than that of the comparative samples during the secondary heating, indicating improved thermal stability of the crystalline regions.

[0138] Table 6 Results of DSC Crystallinity Experiment

[0139] Experimental Example 11 The mechanical properties of the hydrogels prepared in Examples 2-1, 2-4 and Comparative Examples 1 and 4 were tested, and the specific steps are as follows: (1) Testing equipment and fixtures: Shimadzu EZX universal tensile testing machine; alternative ring mold: a transparent mold with an internal confinement cavity. The core component is a cylindrical cavity with a 17G (1.07mm inner diameter) outlet at the bottom. The cavity diameter should be slightly larger than the sample diameter to provide an extrusion channel. Constrained compression fixture: a rigid cylinder with a smooth inner wall and an inner diameter of 20mm, which serves as a confinement ring. Unconstrained control: a standard parallel compression plate is used as a control. The entire testing apparatus is placed in a 37°C constant temperature chamber.

[0140] (2) Extrusion Test: 1) Sample Installation: The cylindrical hydrogel sample (prepared in the same way as in Example 1, except that the mold used is 12 mm in diameter and 5 mm in height) formed by cooling and solidification in the mold was placed into the cavity of the substitute ring, ensuring that the sample was centered; Preloading: A small preload force (5 N) was applied at a low rate (5 N / s) to ensure that the indenter was in full contact with the sample surface and to determine the zero displacement point; 2) Extrusion Test: A compressive load was continuously applied at a loading rate of 25 N / s until the target load of 450 N was reached (this load simulates the pressure inside the intervertebral disc of the human body under specific activities). Throughout the process, the 17G outlet was continuously recorded using a high-definition camera. After reaching 450 N, the load was maintained for 10 seconds, and the results were observed and recorded.

[0141] (3) Compression Test: 1) Sample Preparation: The preparation method is the same as in Experiment 1, except that the mold used has a diameter of 20 mm and a height of 8 mm to ensure that the diameter of the sample is consistent with the inner diameter of the confining ring during the confined test, and that lateral expansion is completely restricted. 2) During the compression test, the sample is tested under both confined and unconfined conditions. Under confined conditions, the sample is placed in the confining ring. Under unconfined conditions, the sample is placed directly on the lower compression plate. Uniaxial compression is performed at a rate of 100% strain / min (for an 8 mm high sample, this is equivalent to a beam speed of 8 mm / min). Compression continues until one of the following two conditions is met: the compressive strain reaches 35% (i.e., the height decreases from 8 mm to 5.2 mm); or the compressive load reaches 400 N.

[0142] (4) Experimental Results: As shown in Table 7, in the extrusion test, the hydrogels of Comparative Example 1 (HG-3) and Comparative Example 4 (HG-4) were both extruded, proving that their mechanical strength was insufficient to withstand such high intervertebral pressure and that there was a risk of displacement. However, the hydrogels of Example 2-1 (HG-5) and Example 2-4 (HG-8) successfully resisted an extrusion pressure of 450N, proving that the hydrogels provided by this invention have excellent extrusion resistance and in-situ reliability. Among them, HG-8 performed the best, with the least deformation.

[0143] As shown in Table 8, in the compression test, the HG-5 and HG-8 hydrogels exhibited superior compressive moduli compared to the comparative examples, regardless of whether they were unconfined or confined. HG-8 consistently showed the highest compressive modulus, which is entirely consistent with the mechanical modulus test results in Experimental Example 1. The confined compressive modulus of the samples in this invention is significantly higher than their unconfined compressive modulus (typically by an order of magnitude). This is because under confined conditions, the material cannot release stress through lateral deformation, resulting in a greatly enhanced compressive strength. This simulates the realistic mechanical environment of a hydrogel tightly encapsulated by surrounding tissues within the human body.

[0144] In terms of load-bearing capacity, HG-5 and HG-8 hydrogels both exhibited greater loads than the comparative sample during compression to 35% strain, demonstrating stronger load-bearing capacity. HG-3 and HG-4 showed relatively weaker load-bearing capacity. Among them, HG-8 demonstrated excellent performance with a load exceeding 400N.

[0145] In summary, the high restricted compressive modulus means that the hydrogel provided by this invention can better resist spinal pressure, prevent intervertebral disc collapse, and provide stable mechanical support. High anti-extrusion properties ensure that the hydrogel provided by this invention remains in place under pressure and does not leak into the spinal canal, endangering nerves. HG-8 demonstrates superior overall performance in both of these key clinical performance indicators. Mechanical property tests strongly demonstrate that the introduction of POx significantly improves the macroscopic mechanical properties of the hydrogel, fundamentally surpassing traditional two-component and PEG-based hydrogels. HG-8 exhibits superior overall performance in all tests: the highest compressive modulus (basic performance), the strongest anti-extrusion capability (can be injected through a 17G needle and withstands pressure of 450N without displacement), and the highest restricted load-bearing capacity (providing stable mechanical support). Therefore, the hydrogel provided by this invention fully meets and even exceeds the key mechanical requirements for biomedical implants such as intervertebral disc replacements.

[0146] Table 7 Extrusion Test Results

[0147] Table 8 Compression Test Results

[0148] Experimental Example 12 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their hydration recovery ability. The specific steps are as follows: (1) Test equipment and materials: TA Instruments Discovery rheometer, equipped with a module and immersion device for dynamic oscillation testing, high-precision electronic balance, sample mold: used to prepare standard-sized cylindrical samples (diameter 20mm, height 8mm), 37℃ constant temperature chamber.

[0149] (2) Solution preparation: High osmotic pressure solution: Accurately weigh 128.2g of polyethylene glycol (PEG, average molecular weight of 20000) and 8.76g of sodium chloride (NaCl), dissolve in deionized water and bring the volume to 1L. This solution is used to simulate the physiological osmotic pressure environment of the human body (approximately 300mOsm / kg).

[0150] (3) Sample preparation and benchmark testing: For each example and comparative example, 20 parallel cylindrical hydrogel samples were prepared using the sample mold described above, following the method in Experimental Example 1. Three samples were randomly selected as the original control group. Then, the initial storage modulus (E'0) was measured using a TA Instruments Discovery rheometer at 37°C, 2% strain, and 1Hz frequency, and the initial wet weight (W0) was recorded. The remaining samples were used for hydration recovery testing.

[0151] (4) Drying treatment: Place the remaining cylindrical hydrogel samples in a constant temperature drying oven at 37°C and dry for 1 hour to simulate the brief drying process during surgical preparation. After drying, take out the samples, weigh their dried weight (Wdry), and immediately proceed to the next hydration test.

[0152] (5) Hydration recovery test: The dried sample is completely immersed in a sufficient amount of high osmotic pressure solution preheated to 37°C. Timing begins. At preset time points (15min, 30min, 1h, 2h, 4h), a sample is taken out of the solution, the surface liquid is gently absorbed with filter paper, and its weight Wt is quickly weighed. The sample is immediately transferred to the DMA test platform and its storage modulus (E') is quickly measured at 37°C with 2% strain and 1Hz frequency.

[0153] (6) Evaluation Indicators: The following indicators were calculated based on the results after immersion for 4 hours: Moisture absorption rate (%) = [(Wt-Wdry) / (W0-Wdry)] × 100%, which reflects the speed at which the hydrogel reabsorbs moisture and restores its initial hydration state. Modulus recovery rate (%) = (E' / E'0) × 100%, which directly quantifies the degree of recovery of the hydrogel's mechanical properties and is the key to evaluating functional recovery.

[0154] (7) Experimental Results: As shown in Table 9, in terms of hydration recovery ability, the hydrogels of Examples 2-1, 2-4, and 2-5 (HG-5, HG-8~9) all exhibited extremely high modulus recovery rates, significantly higher than those of Comparative Example 3 (HG-3) and Comparative Example 4 (HG-4). This demonstrates that the cross-linked network structure of the hydrogel provided by this invention has excellent elastic memory and structural stability. Among them, HG-8 achieved a recovery rate as high as 97.5%, almost completely recovering to its original state, which is highly consistent with the best performance in the mechanical related tests of Examples 1 and 10. The modulus recovery rate of HG-9 was lower than that of HG-8, which is consistent with the phenomenon of over-crosslinking that may be caused by excessive POx observed in Example 8, but its performance is still far superior to that of the comparative examples.

[0155] In terms of hydration recovery rate, HG-8 exhibits the fastest recovery speed, indicating that although its network structure is dense and strong, it possesses ideal hydrophilic channels, allowing water to penetrate rapidly and uniformly, thus enabling the material to quickly expand and recover its shape. Hydrogels containing POx (HG-5~HG-11) all show significantly faster recovery rates than HG-4, suggesting that the introduction of POx did not sacrifice the material's hydrophilicity; on the contrary, it may have optimized the transport pathways of water molecules within the polymer network. HG-4 has the slowest hydration recovery rate and a lower final modulus recovery rate, which may stem from phase separation or changes in crystal morphology of its PEG component during the drying-rehydration process, resulting in the network structure not being fully restored.

[0156] Table 9 Results of Hydration Recovery Capacity Experiment

[0157] Experimental Example 13 The hydrogels prepared in Examples 2-1 to 2-7 and Comparative Examples 1-4 were tested for their leakage performance. The specific steps are as follows: The obtained hydrogel was filled into a 5mL syringe, cooled, and solidified to obtain a solidified hydrogel with a volume of 5.0mL. The syringe containing the hydrogel was preheated to 65°C, and a 15G injection needle was used to attempt to eject it. The results showed that the solidified hydrogels of each example and comparative example could be ejected smoothly. The ejection time of 1mL dose is shown in Table 10 below. There was no leakage at the needle tip when the hydrogels of examples HG-5 to HG-11 were ejected. Among them, the ejection time of HG-5 to HG-8 was between 20-29s, which is more conducive to clinicians to inject viscous materials with one hand during surgery, without splashing due to being too fast or causing surgical fatigue due to being too slow. The comparative examples (HG-1 to HG-4) generally have simulated injection pressures exceeding 140 kPa (as shown in Table 4). When manually injecting, the large power consumption caused by friction and internal viscosity is required, which significantly prolongs the time. This can easily cause needle leakage during surgery. The test results show that obvious leakage was observed in HG-2 and HG-3. Although no leakage was observed in HG-1 and HG-4, they have a high risk of leakage.

[0158] Table 10 Leakage Performance Test Results

[0159] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hydrogel composition, characterized in that, By weight, it includes the following raw materials: The mixture comprises 10-35 parts of oxazoline polymer, 10-20 parts of polyvinyl alcohol, 0.1-1.5 parts of polyvinylpyrrolidone, and 40-75 parts of water; the oxazoline polymer has the structure shown in formula (I): Equation (I); Wherein, R1 is selected from unsubstituted or hydroxylated C1-C22 straight-chain alkyl groups, unsubstituted or hydroxylated C3-C22 branched alkyl groups, and unsubstituted or hydroxylated C3-C22 cycloalkyl groups. or One or more of the following; n represents the number of repeating units, an integer selected from 1 to 150, and m represents the number of repeating units, an integer selected from 1 to 100.

2. The hydrogel composition according to claim 1, characterized in that, R1 is selected from unsubstituted or hydroxylated C1-C10 straight-chain alkyl groups, unsubstituted or hydroxylated C3-C10 branched-chain alkyl groups, and unsubstituted or hydroxylated C3-C22 cycloalkyl groups. or One or more of them.

3. The hydrogel composition according to claim 1, characterized in that, R1 is selected from unsubstituted or hydroxylated methyl, unsubstituted or hydroxylated ethyl, unsubstituted or hydroxylated n-propyl, unsubstituted or hydroxylated isopropyl, unsubstituted or hydroxylated n-butyl, unsubstituted or hydroxylated isobutyl, unsubstituted or hydroxylated n-pentyl, unsubstituted or hydroxylated n-hexyl, , , One or more of them.

4. The hydrogel composition according to claim 1, characterized in that, R1 is selected from methyl, , , , One or more of them.

5. The hydrogel composition according to claim 1, characterized in that, The oxazoline polymer has the following structure: 。 6. The hydrogel composition according to claim 1, characterized in that, By weight, it comprises the following raw materials: 10-30 parts of oxazoline polymer, 10-15 parts of polyvinyl alcohol, 0.1-1 parts of polyvinylpyrrolidone, and 49-75 parts of water.

7. The hydrogel composition according to claim 1 or 6, characterized in that, The polyvinyl alcohol has an average molecular weight of 1 million to 1.5 million and a degree of hydrolysis ≥99%. And / or, the K value of the polyvinylpyrrolidone is 27-32; And / or, the average molecular weight of the oxazoline polymer is 3000-30000; And / or, the hydrogel composition further includes 4-6 parts by weight of contrast agent.

8. The hydrogel composition according to claim 7, characterized in that, The contrast agent includes at least one of barium sulfate, iohexol, iopamidol, iodixanol, iopromide, gadopentetate dimeglumine, gadotate dimeglumine, and gadobutrol.

9. A method for preparing the hydrogel composition according to any one of claims 1-8, characterized in that, Includes the following steps: Step S1: Mix polyvinyl alcohol, polyvinylpyrrolidone and a portion of water, and then heat the mixture to obtain solution A; Step S2: The oxazoline polymer is mixed with another portion of water and then heated a second time to obtain solution B; Step S3: Mix solution A with solution B and allow to stand to obtain a hydrogel; Alternatively, it may include the following steps: Step S11: Mix polyvinyl alcohol, polyvinylpyrrolidone and water and then heat for the first time to obtain solution A; Step S12: The oxazoline polymer is subjected to a second heating to obtain a melt; Step S13: Mix solution A with the melt, and allow it to stand to obtain a hydrogel; When the hydrogel contains a contrast agent, steps S1 and S11 further include the step of adding the contrast agent to solution A.

10. The method for preparing the hydrogel composition according to claim 9, characterized in that, The preparation method of the oxazoline polymer includes the following steps: polymerizing a compound with the structure shown in formula (II) with 2-ethyl-2-azoline to obtain oxazoline polymer E; Alternatively, the preparation method of the oxazoline polymer includes the following steps: hydrolyzing oxazoline polymer E to obtain a polymer with the structure shown in formula (III); esterifying the polymer with the structure shown in formula (III) with R1-OH to obtain an oxazoline polymer; wherein R1 is as defined in any of claims 1-5; Oxazoline polymer E; Formula (II); Formula (III).

11. The method for preparing the hydrogel composition according to claim 10, characterized in that, The R1-OH is selected from one or more of glycerol, n-pentanol, xylitol, and inositol.

12. The method for preparing the hydrogel composition according to claim 9, characterized in that, The mass ratio of a portion of the water in step S1 to another portion of the water in step S2 is 1-2:1; And / or, in steps S1 and S11, the temperature of the first heating is 90-100°C; And / or, in steps S2 and S12, the temperature of the second heating is 75-85°C; And / or, in steps S3 and S13, the mixing time is 3-7 minutes; And / or, in steps S3 and S13, the settling temperature is 20-30°C and the time is 1-3 hours.

13. The use of the hydrogel composition according to any one of claims 1-8 or the hydrogel composition prepared by the method of any one of claims 9-12 in the preparation of tissue-engineered biomaterials and / or drug carriers.

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