A self-gelling hyaluronic acid-based physical cross-linking hydrogel, and a preparation method and application thereof

Abstract

This invention provides a method for preparing and applying a self-gelling hyaluronic acid-based physically crosslinked hydrogel. Hyaluronic acid, dihydrazide carbonate, and deionized water are mixed and dissolved, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole. The reaction is followed by dialyzing and lyophilization to obtain hydrazide-modified hyaluronic acid. The hydrazide-modified hyaluronic acid and the thymine derivative thymine-1-acetic acid are mixed and dissolved, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The pH is adjusted, and the reaction is followed by dialyzing and lyophilization to obtain thymine-modified hyaluronic acid. The thymine-modified hyaluronic acid is dissolved in PBS buffer, homogenized, and allowed to stand to obtain the final product. The thymine-grafted hyaluronic acid material utilizes the property of nucleic acid bases to spontaneously form multiple hydrogen bonds, achieving hydrogen bond-driven self-gelling. Physical crosslinking retains the good hydration ability of hyaluronic acid and imparts injectability and adhesiveness to the gel.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically to a self-gelling hyaluronic acid-based physically crosslinked hydrogel for intervertebral disc regeneration and its preparation method. Background Technology

[0002] Discectomy and disc fusion have long been considered the gold standard for treating degenerative intervertebral discs. However, the biomechanical instability of the intervertebral disc following these procedures can cause stress concentration in the annulus fibrosus or adjacent vertebrae, leading to accelerated degeneration of the damaged disc, re-protrusion of the nucleus pulposus, or herniation of an adjacent disc. Restoring the biological function of the damaged intervertebral disc remains a clinical challenge.

[0003] Due to the excellent hydration capacity of hyaluronic acid, similar to that of nucleus pulposus tissue, hyaluronic acid-based hydrogels have been widely used in the treatment of degenerative intervertebral discs. However, regrettably, although various hyaluronic acid-based hydrogels have been constructed, most are unsuitable for nucleus pulposus regeneration. For example, UV-crosslinked hydrogels prepared from methacryloyl hyaluronic acid (HAMA) are fragile and difficult to inject with a syringe; aldehyde-modified hyaluronic acid (HA-ADH) can form hydrogels via the Schiff base reaction and exhibits good tissue adhesion, but excessive aldehyde groups may induce cytotoxicity and biological side effects. Furthermore, hyaluronic acid physical gels can be prepared in an acidic environment through freeze-thaw cycles, but the application of hyaluronic acid cryophysical gels in cell delivery is limited due to the harsh preparation conditions.

[0004] Currently, research on cell therapy using hyaluronic acid solution-loaded mesenchymal stem cells to treat degenerative intervertebral discs has entered the clinical research stage. However, the anodic apoptosis and low cell survival rate of implanted exogenous stem cells pose significant challenges to the clinical efficacy of treatment for degenerative intervertebral discs. In the early stages of intervertebral disc degeneration, adverse microenvironmental factors such as inflammation, hypoxia, and acidity can further induce cell damage, such as programmed cell death, exacerbation of inflammation, and nerve ingrowth, further accelerating the degenerative process. Furthermore, the liquid state of the hyaluronic acid solution carrier inevitably leads to cell leakage, resulting in osteophyte formation.

[0005] Therefore, for the treatment of degenerative intervertebral discs, it is crucial to design a cell delivery system that can modulate the microenvironment of the degenerated site and ensure the viability of the supplemented exogenous cells. Furthermore, to ensure that the encapsulated cells are accurately deposited at the implantation site, the delivery system also needs to possess appropriate viscoelasticity and adhesiveness. Summary of the Invention

[0006] This invention overcomes the shortcomings of existing technologies, such as poor hydration capacity, low bioactivity, and difficulty in adapting to the microenvironment of degenerated intervertebral discs, thus hindering the preservation of exogenous cell activity and the restoration of intervertebral disc biomechanical function. This invention provides a self-gelling hyaluronic acid-based physically crosslinked hydrogel, its preparation method, and its applications. The preparation method is simple, achieving hydrogen-bonded self-gelling of hyaluronic acid physical gel by grafting thymine onto hyaluronic acid. Physical crosslinking preserves the excellent hydration capacity of hyaluronic acid and imparts injectability and adhesiveness to the gel. Doping with inorganic manganese dioxide nanoparticles improves the gel's adaptability to harsh environments. The intervertebral disc regeneration scaffold (HATMn-MSCs) prepared by this method can effectively improve the survival rate of supplemented exogenous stem cells and restore the biomechanical function of the intervertebral disc. Preliminary animal experiments have confirmed its application potential in intervertebral disc regeneration.

[0007] The objective of this invention is achieved through the following technical solution.

[0008] A self-gelling hyaluronic acid-based physically crosslinked hydrogel and its preparation method are carried out according to the following steps:

[0009] Step 1: Dissolve sodium hyaluronate powder (HA) in ultrapure water. After it is completely dissolved, add dihydrazide carbonate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 1-hydroxybenzotriazole (HOBT) in sequence. After reacting for 24-96 hours, transfer the reaction solution to a dialysis bag and lyophilize by dialyzing to obtain hydrazide-modified hyaluronic acid (HA-HYD).

[0010] The molecular weight of hyaluronic acid is 2-10w, the concentration of hyaluronic acid in the reaction system is 5‰-15‰, and the molar ratio of dihydrazide carbonate, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBT) is (1-5):(1-3):1:2.

[0011] Step 2: Dissolve the hyaluronic acid (HA-HYD) prepared in Step 1 in ultrapure water. After it is completely dissolved, add thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) in sequence. Adjust the pH of the above reaction system to 4-7. After reacting for 12-72 hours, transfer the reaction solution to a dialysis bag, dialyze and freeze dry to obtain thymine-grafted hyaluronic acid (HAT).

[0012] The concentration of hydrazide-modified hyaluronic acid in the reaction system is 5‰-2%, and the mass ratio of hydrazide-modified hyaluronic acid, thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) is (1-5):(1-3):1:1;

[0013] Step 3: Dissolve the thymine-modified hyaluronic acid prepared in Step 2 in PBS buffer, vortex homogenize, and let stand at room temperature to obtain thymine-hyaluronic acid hydrogel (HAT), which is a self-gelling hyaluronic acid-based physically cross-linked hydrogel.

[0014] The concentration of thymine-hyaluronic acid is 1-20%.

[0015] In step 1, the molecular weight of hyaluronic acid (HA) is 5w, the concentration of hyaluronic acid in the reaction system is 8‰, and the molar ratio of dihydrazide carbonate, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBT) is 2:1:1:2. The reaction is carried out at 25°C for 48h.

[0016] In step 2, the concentration of hydrazide-modified hyaluronic acid in the reaction system is 1%, the reaction is carried out at 25°C for 24 hours, the pH of the reaction system is 4.75, and the mass ratio of hydrazide-modified hyaluronic acid, thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) is 2:1:0.1:0.1.

[0017] In step 3, the concentration of thymine-hyaluronic acid is 5%.

[0018] Among them, thymine-hyaluronic acid (HAT) can spontaneously form physical gels through hydrogen bonding without cross-linking agents; the storage modulus of the gel is 40-2000 Pa; the hydration capacity of 5% thymine-hyaluronic acid (HAT) hydrogel at osmotic pressures of 0.3, 1.1, 2.5, 4.4, and 6.9 atm is 20.79±2.76, 17.28±1.36, 7.07±0.29, 4.73±0.07, and 3.78±0.31 g H2O / g, respectively, and the adhesion strength is 1-3 kPa.

[0019] A self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold (HAT-MSCs) is formed by combining the self-gelling hyaluronic acid-based physically cross-linked hydrogel prepared above with bone marrow mesenchymal stem cells.

[0020] A manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel and its preparation method are disclosed. In step 3, the thymine-modified hyaluronic acid prepared in step 2 is dissolved in PBS buffer containing manganese dioxide, homogenized by vortexing, and allowed to stand at room temperature to obtain manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn), which is a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel.

[0021] In the PBS buffer containing dispersed manganese dioxide, the concentration of manganese dioxide is 100 μg / ml-500 μg / ml.

[0022] In step 3, the concentration of manganese dioxide in the PBS buffer containing manganese dioxide is 200 μg / ml.

[0023] Manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffolds were prepared by combining the manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogels prepared above with bone marrow mesenchymal stem cells to form manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffolds (HATMn-MSCs).

[0024] Applications of self-gelling hyaluronic acid-based physically cross-linked hydrogels, self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffolds, manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogels, or manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffolds in biomedical materials, especially medical materials for intervertebral disc regeneration.

[0025] The beneficial effects of this invention are as follows: The preparation method of this invention is simple. A hydrogen-bonded, self-gelling hyaluronic acid physical gel is achieved by grafting thymine onto hyaluronic acid. Physical cross-linking preserves the gel's hydration capacity and imparts injectability and adhesiveness. Doping with inorganic nanoparticles (manganese dioxide) enhances the gel's adaptability to harsh environments. The intervertebral disc regeneration scaffold (HATMn-MSCs) prepared by this method can effectively improve the survival rate of supplemented exogenous stem cells and restore the biomechanical function of the intervertebral disc. Preliminary animal experiments have confirmed its potential application in intervertebral disc regeneration. Attached Figure Description

[0026] Figure 1 These are NMR images of hydrazide-substituted hyaluronic acid, thymine-1-acetic acid, and thymine-hyaluronic acid (HAT).

[0027] Figure 2 These are Fourier transform infrared spectra of hydrazide-substituted hyaluronic acid, thymine-1-acetic acid, and thymine-hyaluronic acid (HAT).

[0028] Figure 3This is a characterization of the basic properties of the gel in this invention, wherein (a) is a comparison of the states of hyaluronic acid, hydrazide-modified hyaluronic acid, and thymine-hyaluronic acid in PBS; (b) is a rheological diagram of hyaluronic acid, hydrazide-modified hyaluronic acid, and thymine-hyaluronic acid gels in time-scan mode; (c) is a shear-thinning curve and injectability diagram of thymine-hyaluronic acid gel; and (d) is a schematic diagram of the self-healing properties of thymine-hyaluronic acid gel.

[0029] Figure 4 This invention characterizes the adhesive properties of thymine-hyaluronic acid, where (a) is a schematic diagram of the adhesion of HAT gel to iron, ceramics, wood, glass, plastics, polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET); (b) is a schematic diagram of the adhesion of HAT gel to the small intestine, bone, skin, heart, and liver; and (c) is a characterization diagram of the overlap shear adhesion force of HAT gel to pigskin.

[0030] Figure 5 This invention describes the performance of HATMn, which is doped with MnO2 nanoparticles, in scavenging ROS. (a) is a statistical graph of HATMn's efficiency in scavenging hydrogen peroxide; (b) is a statistical graph of HATMn's efficiency in scavenging hydroxyl radicals; and (c) is the live and dead staining of MSCs under the following conditions: control culture medium, hydrogen peroxide treatment (H2O2), and treatment with both HATMn and hydrogen peroxide (HATMn+H2O2).

[0031] Figure 6 These are the results of hematoxylin-eosin staining (a) and alicin blue staining (b) at different time points in the treatment of rat caudal intervertebral disc degeneration using HATMn-MSCs in this invention. Detailed Implementation

[0032] The technical solution of the present invention will be further described below through specific embodiments.

[0033] Example 1

[0034] Preparation of thymine-hyaluronic acid (HAT) gel:

[0035] Step 1: First, weigh 1g of HA (Mw = 5w) into a 250mL round-bottom flask, add 150mL of ultrapure water as a solvent and stir until the HA is completely dissolved. Then add 0.2g of dihydrazide carbonate and stir until completely dissolved. Next, add 0.5g of 1-hydroxybenzotriazole (HOBT) and 0.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4, and react at 25℃ for 24h. After the reaction is complete, dialyze in 0.1M NaCl solution for two days, dialyze in pure water for one day, and freeze-dry to obtain hydrazide-modified hyaluronic acid (HA-HYD).

[0036] Step 2: Preparation of thymine-hyaluronic acid (HAT): First, weigh 0.5 g of HA-HYD into a 250 mL round-bottom flask, add 100 mL of ultrapure water as a solvent and stir until HA-HYD is completely dissolved. Then add 0.5 g of thymine-1-acetic acid and stir until completely dissolved. Next, add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4.75, and react at 25 °C for 12 h. After the reaction is complete, dialyze in 0.1 M NaCl solution for two days, dialyze in pure water for one day, and lyophilize to obtain thymine-hyaluronic acid (HAT).

[0037] Step 3: Weigh 0.05g of HAT into a centrifuge tube using an analytical balance, add 1ml of ultrapure water, vortex and appropriately increase the temperature to accelerate the uniform mixing of HAT in PBS, and then let it stand at room temperature for 2h to obtain thymine-hyaluronic acid hydrogel (HAT), which is a self-gelling hyaluronic acid-based physically cross-linked hydrogel.

[0038] Example 2

[0039] Preparation of thymine-hyaluronic acid (HAT) gel:

[0040] Step 1: First, weigh 1.22 g of HA (Mw = 5w) into a 250 mL round-bottom flask, add 150 mL of ultrapure water as a solvent and stir until the HA is completely dissolved. Then add 0.54 g of dihydrazide carbonate and stir until completely dissolved. Next, add 0.81 g of 1-hydroxybenzotriazole (HOBT) and 0.54 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4.8, and react at 25 °C for 48 h. After the reaction is complete, dialyze against 0.1 M NaCl solution for two days, then against pure water for one day, and freeze-dry to obtain hydrazide-modified hyaluronic acid (HA-HYD).

[0041] Step 2: Preparation of thymine-hyaluronic acid (HAT): First, weigh 1g of HA-HYD into a 250mL round-bottom flask, add 100mL of ultrapure water as a solvent and stir until HA-HYD is completely dissolved. Then add 0.6g of thymine-1-acetic acid and stir until completely dissolved. Next, add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4.75, and react at 25℃ for 24h. After the reaction is complete, dialyze in 0.1M NaCl solution for two days, dialyze in pure water for one day, and freeze-dry to obtain thymine-hyaluronic acid (HAT).

[0042] Step 3: Weigh 0.1g of HAT using an analytical balance and place it in a centrifuge tube. Add 1ml of ultrapure water, vortex and appropriately increase the temperature to accelerate the uniform mixing of HAT in PBS. Then let it stand at room temperature for 2 hours to obtain thymine-hyaluronic acid hydrogel (HAT), which is a self-gelling hyaluronic acid-based physically cross-linked hydrogel.

[0043] Example 3

[0044] Preparation of thymine-hyaluronic acid (HAT) gel:

[0045] Step 1: First, weigh 3g of HA (Mw = 5w) into a 250mL round-bottom flask, add 150mL of ultrapure water as a solvent and stir until the HA is completely dissolved. Then add 1g of dihydrazide carbonate and stir until completely dissolved. Next, add 1g of 1-hydroxybenzotriazole (HOBT) and 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 7, and react at 25℃ for 72h. After the reaction is complete, dialyze in 0.1M NaCl solution for two days, dialyze in pure water for one day, and freeze-dry to obtain hydrazide-modified hyaluronic acid (HA-HYD).

[0046] Step 2: Preparation of thymine-hyaluronic acid (HAT): First, weigh 2g of HA-HYD into a 250mL round-bottom flask, add 100mL of ultrapure water as a solvent and stir until HA-HYD is completely dissolved. Then add 1g of thymine-1-acetic acid and stir until completely dissolved. Next, add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4.75, and react at 25℃ for 48h. After the reaction is complete, dialyze in 0.1M NaCl solution for two days, dialyze in pure water for one day, and lyophilize to obtain thymine-hyaluronic acid (HAT).

[0047] Step 3: Weigh 0.15g of HAT using an analytical balance and place it in a centrifuge tube. Add 1ml of ultrapure water, vortex and appropriately increase the temperature to accelerate the uniform mixing of HAT in PBS. Then let it stand at room temperature for 2h to obtain thymine-hyaluronic acid hydrogel (HAT), which is a self-gelling hyaluronic acid-based physically cross-linked hydrogel.

[0048] Example 4:

[0049] Weigh 1 mg of manganese dioxide (MnO2) using an analytical balance and place it in a 15 ml centrifuge tube. Add 10 ml of PBS and sonicate to disperse the manganese dioxide (MnO2) evenly. Weigh 0.02 g of HAT prepared in step 2 of Example 1 using an analytical balance and place it in a centrifuge tube. Add 1 ml of PBS containing dispersed manganese dioxide (MnO2) and vortex to mix the HAT evenly in the PBS containing dispersed manganese dioxide. Then let it stand at room temperature for 2 h to obtain manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn), which is a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel.

[0050] Example 5:

[0051] Weigh 2 mg of manganese dioxide (MnO2) using an analytical balance and place it in a 15 ml centrifuge tube. Add 10 ml of PBS and sonicate to disperse the manganese dioxide (MnO2) evenly. Weigh 0.05 g of HAT prepared in step 2 of Example 2 using an analytical balance and place it in a centrifuge tube. Add 1 ml of PBS containing dispersed manganese dioxide (MnO2) and vortex to mix the HAT evenly in the PBS containing dispersed manganese dioxide. Then let it stand at room temperature for 2 h to obtain manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn), which is a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel.

[0052] Example 6:

[0053] Weigh 5 mg of manganese dioxide (MnO2) using an analytical balance and place it in a 15 ml centrifuge tube. Add 10 ml of PBS and sonicate to disperse the manganese dioxide (MnO2) evenly. Weigh 0.15 g of HAT prepared in step 2 of Example 3 using an analytical balance and place it in a centrifuge tube. Add 1 ml of PBS containing dispersed manganese dioxide (MnO2) and vortex to mix the HAT evenly in the PBS containing dispersed manganese dioxide. Then let it stand at room temperature for 2 h to obtain manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn), which is a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel.

[0054] NMR and IR characterization of HAT gels: such as Figure 1 As shown, in thymine-1-acetic acid 1The 1H NMR spectrum clearly shows signals at 1.86, 4.51, and 7.4 ppm, corresponding to the hydrogen atoms of the methyl (a), methylene (b), and alkene (c) groups in thymine-1-acetic acid, respectively. Comparison of the NMR spectra of HA and HAT confirms the successful synthesis of HAT. In the HAT spectrum, besides the sugar ring structure of HA visible at 3-4 ppm, the characteristic absorption peak of the alkene at 7.43 ppm and the methyl proton absorption peak at 1.86 ppm both originate from the coupling of thymine. Figure 2 The results showed that FTIR analysis clarified the hydrogen-bonded gelation mechanism of the HAT gel. Compared with HA, the gel at 1635 cm⁻¹... -1 and 1560cm -1 The slight red shift of the symmetric stretching vibration and amide II band vibration peaks corresponding to C=O in HAT indicates the formation of hydrogen bonds between C=O groups.

[0055] Hydration capacity and swelling behavior test of HAT gel: First, solutions with different osmotic pressures (0.03-0.69 MPa) were prepared using PEG (MW = 20,000). The mass of the lyophilized HAT was recorded as W0 and placed in a dialysis tube (8,000 MWCO). The dialysis tube was then immersed in PEG solutions with different osmotic pressures for 24 hours. The mass of the hydrated gel was recorded again as W1. The hydration capacity of HAT was calculated using the following formula:

[0056]

[0057] The swelling behavior of the gel was determined by immersing the lyophilized gel in PBS at 37°C, weighing the gel again at regular intervals, and calculating the increase in mass ratio. Figure 3 As shown, HAT gels exhibit good hydration capabilities, with hydration capacities of 20.79±2.76, 17.28±1.36, 7.07±0.29, 4.73±0.07, and 3.78±0.31 g H2O / g at 0.3 atm, 1.1 atm, 2.5 atm, 4.4 atm, and 6.9 atm, respectively. Furthermore, swelling results indicate that 5% HAT can swell to 22 times its weight, and 15% HAT can swell to 45 times its original weight. This increase in gel swelling with increasing solid content can be attributed to the increased structural stability of the gel with increasing solid content, resulting in a longer time required for structural collapse (the weight of the 5% HAT hydrogel decreased after 58.5 hours, 10% HAT after 84 hours, and 15% HAT after 116 hours), thus leading to a higher swelling rate in PBS.

[0058] Rheological property testing of HAT gel: The gel state of HAT was determined using time-scan mode. For example... Figure 3As shown, the storage modulus of HAT gels is greater than their loss modulus, indicating that HAT is in a gel state. In contrast, HA-HYD gels exhibit solution behavior where the loss modulus is greater than the storage modulus. Oscillating mode variable strain scanning rheological spectra show that HAT has good shear-thinning behavior and can be easily extruded from the syringe. Due to the large number of hydrogen bonds in HAT gels, HAT also exhibits excellent self-healing properties. When two gels are gently placed together, they heal into a single unit within one minute, capable of withstanding tensile and torsional deformations.

[0059] Characterization of the adhesion behavior of HAT gel: First, a 10% HAT gel was prepared, and then the HAT gel was interacted with different materials to examine the adhesion between the gel and the materials. Figure 4 As shown, HAT gel exhibits adhesive properties to a variety of materials, including iron, ceramics, wood, glass, plastics, polytetrafluoroethylene (PTFE), polyethylene (PE), polyethylene terephthalate (PET), and other organic and inorganic materials, as well as tissues such as the small intestine, bone, skin, heart, and liver. Using pigskin as an adhesion model, the adhesion strength of the gel to pigskin was quantitatively characterized using an overlap shear test. The test results show that the adhesion strength gradually increases with the increase of the gel's solid content. A gel with a solid content of 15% can achieve an adhesion strength of 3.5 kPa to pigskin. Furthermore, the adhesion failure of the gel is mainly caused by damage to the gel matrix.

[0060] H2O2 and OH· scavenging ability test of HATMn gel: MnO2 nanoparticles were doped into HAT gels at concentrations of 200 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml, and 1000 μg / ml, and the scavenging ability of HATMn for H2O2 and OH· was measured. First, a Ti(SO4)2 solution was prepared, and 0.2 M H2O2 and HATMn were co-incubated for 10 min. Then, 2.6 ml of Ti(SO4)2 solution was added to detect residual H2O2. The absorbance of the supernatant at 405 nm was then measured using a microplate reader. For the determination of OH· scavenging ability, a solution of 9 mM FeSO4, 8.8 mM H2O2, and 9 mM salicylic acid was prepared. Then, 200 μl of 9 mM FeSO4 and 200 μl of 8.8 mM H2O2 were mixed to generate OH·. Subsequently, HATMN was added and incubated at 37°C for 30 minutes. Finally, 200 μl of 9 mM salicylic acid was added, and the absorbance of the supernatant at 510 nm was measured using a microplate reader. Figure 5As shown, the free radical scavenging ability of HATMn increases with increasing MnO2 concentration. When the MnO2 concentration is 200 μg / ml, the H2O2 scavenging ability of HATMn reaches 70%, while when the MnO2 concentration is 400 μg / ml, the H2O2 scavenging ability of HATMn reaches almost 100%. When the MnO2 concentration is 1000 μg / ml, the OH· scavenging ability of HATMn reaches 70%.

[0061] Test of the ability of HATMn gel to protect cells from H2O2 damage: First, mesenchymal stem cells were placed in a solution of 1*10... 5 Cells were seeded in 12-well plates and cultured for 24 hours. The medium was then replaced with conditioned medium containing 400 μM H₂O₂, and HAT Mn gel was added to the wells. After 4 hours of culture, cells were stained with a live / dead cell staining agent (Calcein-AM / PI), and finally photographed using an inverted fluorescence microscope. Figure 6 As shown, in the group without HATMn gel, many cells showed PI positivity, indicating that the cell state was damaged. In contrast, in the HATMn group, almost all cells showed AM positivity and PI negativity, indicating that the cell state was good. This proves that HATMn can effectively protect cells from H2O2 damage.

[0062] The manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel prepared in Example 5 was combined with bone marrow mesenchymal stem cells to form manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffolds (HATMn-MSCs).

[0063] Evaluation of the therapeutic effect of HATMn-MSCs scaffolds on rat caudal intervertebral disc degeneration: First, a rat caudal intervertebral disc degeneration model was established and allowed 4 weeks for successful model establishment. At week 5, HA-MSCs or HATMn-MSCs were injected into the nucleus pulposus using a microinjector; the corresponding intervertebral discs were designated as the HA-MSCs group and HATMn-MSCs group, respectively. As controls, only punctured discs and normal discs were also included, designated as the IVDD group and normal group, respectively. At weeks 4, 8, and 12 after administration of HA-MSCs and HATMn-MSCs, samples were harvested for fixation, decalcification, and hematoxylin-eosin and alicin blue staining. Figure 6As shown in Figure a, hematoxylin-eosin (HE) staining images indicate that, compared to normal IVD, the nucleus pulposus (NP) content in the IVDD group gradually decreased over time, and the boundary between the NP and annulus fibrosus was blurred in the IVD at 12 weeks. In the HA-MSCs group, NP continued to degenerate, leading to AF rupture and invasion of the NP space. Therefore, the residual amount of NP was small at 12 weeks. In contrast, in the HATTMn-MSCs group, a clear boundary between NP and AF was observed at each time point. The fibrous tissue in the AF was regularly arranged. Unlike the inflammatory cell aggregation in the IVD region of the IVDD and HA-MSCs groups, the HATTMn-MSCs group showed a smaller inflammatory response. Figure 6 As shown in Figure b, alexandrite blue staining revealed that in the normal group, IVD showed significant proteoglycan staining in the NP region, while the AF region exhibited good collagen arrangement and minimal cell presence. In contrast, alexandrite blue staining was not prominent in the IVDD group. Even after HA-MSCs treatment, proteoglycan staining outside the AF region and NP heterogeneity remained increased. In comparison, proteoglycans and collagen in the HATMn-MSCs group showed a more regular distribution within the intervertebral disc. Furthermore, we observed well-organized collagen arrangement, exhibiting a typical natural morphology, similar to a natural intervertebral disc. In conclusion, these results further validate the effectiveness of the HATMn-MSCs strategy in delaying IVDD progression and promoting NP regeneration.

[0064] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a self-gelling hyaluronic acid-based physically crosslinked hydrogel, characterized in that: The following steps are followed: Step 1, dissolve sodium hyaluronate powder in ultrapure water. After it is completely dissolved, add dihydrazide carbonate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole in sequence. After reacting for 24-96 hours, transfer the reaction solution to a dialysis bag and lyophilize by dialyzing to obtain hydrazide-modified hyaluronic acid. The molecular weight of sodium hyaluronate is 2-10w, the concentration of sodium hyaluronate in the reaction system is 5‰-15‰, and the molar ratio of dihydrazide carbonate, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is (1-5):(1-3):1:

2. Step 2: Dissolve the hydrazide-modified hyaluronic acid obtained in Step 1 in ultrapure water. After complete dissolution, add thymidine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide in sequence, and adjust the pH of the reaction system to 4-7. After reacting for 12-72 hours, transfer the reaction solution to a dialysis bag, dialyze, and freeze-dry to obtain thymidine-grafted hyaluronic acid. The concentration of hydrazide-modified hyaluronic acid in the reaction system is 5‰-2%, and the mass ratio of hydrazide-modified hyaluronic acid, thymidine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is (1-5):(1-3):1:

1. Step 3: Dissolve the thymine-grafted hyaluronic acid obtained in Step 2 in PBS buffer, vortex homogenize, and let stand at room temperature to obtain thymine-hyaluronic acid hydrogel, i.e., self-gelling hyaluronic acid-based physically cross-linked hydrogel; wherein, the concentration of thymine-hyaluronic acid is 1-20%.

2. The method for preparing a self-gelling hyaluronic acid-based physically crosslinked hydrogel according to claim 1, characterized in that: In step 1, the molecular weight of sodium hyaluronate is 5w, the concentration of sodium hyaluronate in the reaction system is 8‰, and the molar ratio of dihydrazide carbonate, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 2:1:1:

2. The reaction is carried out at 25°C for 48 h. In step 2, the concentration of hydrazide-modified hyaluronic acid in the reaction system is 1%, the reaction is carried out at 25°C for 24 h, and the pH of the reaction system is 4.

75. In step 3, the concentration of thymine-hyaluronic acid is 5%.

3. A self-gelling hyaluronic acid-based physically crosslinked hydrogel is prepared using the preparation method described in claim 1 or 2.

4. A self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold, characterized in that: A self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold is formed by combining it with bone marrow mesenchymal stem cells as described in claim 3.

5. The application of the self-gelling hyaluronic acid-based physically crosslinked hydrogel as described in claim 3 in the preparation of biomedical materials.

6. The application according to claim 5, characterized in that, The biomedical material is a medical material used for intervertebral disc regeneration.

7. The application of the self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold as described in claim 4 in the preparation of biomedical materials.

8. The application according to claim 7, characterized in that, The biomedical material is a medical material used for intervertebral disc regeneration.

9. A method for preparing a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel based on the preparation method of claim 1 or 2, characterized in that: In step 3, the thymine-grafted hyaluronic acid prepared in step 2 is dissolved in PBS buffer containing manganese dioxide, vortexed for homogenization, and allowed to stand at room temperature to obtain manganese dioxide nanoparticle-doped thymine-hyaluronic acid hydrogel, i.e., manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel; wherein, the concentration of manganese dioxide in the PBS buffer containing manganese dioxide is 100 μg / ml-500 μg / ml.

10. The method for preparing a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel according to claim 9, characterized in that: In step 3, the concentration of manganese dioxide in the PBS buffer containing manganese dioxide is 200 μg / ml.

11. A manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel prepared by the preparation method of claim 9 or 10.

12. A manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold, characterized in that: A manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold is formed by combining it with bone marrow mesenchymal stem cells as described in claim 11.

13. The application of the manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel as described in claim 11 in the preparation of biomedical materials.

14. The application according to claim 13, characterized in that, The biomedical material is a medical material used for intervertebral disc regeneration.

15. The application of the manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold as described in claim 12 in the preparation of biomedical materials.

16. The application according to claim 15, characterized in that, The biomedical material is a medical material used for intervertebral disc regeneration.

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