Self-gelling hyaluronic acid-based physical crosslinking hydrogel as well as preparation method and application thereof

Abstract

The invention provides a preparation method and application of self-gelling hyaluronic acid-based physical crosslinking hydrogel, hyaluronic acid, carbonic acid dihydrazide and deionized water are mixed and dissolved, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole are added, a reaction is carried out, dialysis and freeze-drying are carried out, and hydrazide hyaluronic acid is obtained; the preparation method comprises the following steps: mixing and dissolving hydrazide hyaluronic acid and thymine derivative thymidine-1 acetic acid, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH value, reacting, dialyzing and freeze-drying to obtain thymine modified hyaluronic acid; dissolving the thymine modified hyaluronic acid in a PBS buffer solution, homogenizing, and standing to obtain the thymine modified hyaluronic acid. Due to the property that nucleic acid bases can spontaneously form multiple hydrogen bonds, the prepared thymine grafted hyaluronic acid material realizes hydrogen bond driven self-gelation. Physical crosslinking retains good hydration ability of hyaluronic acid and endows the gel with injectability and adhesion.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and more particularly to a self-gelling hyaluronic acid-based physically crosslinked hydrogel for intervertebral disc regeneration and a preparation method thereof. Background Art

[0002] Nucleotomy and intervertebral disc fusion have always been regarded as the gold standard for the treatment of degenerative intervertebral discs. However, the biomechanical instability of the intervertebral disc after nucleotomy and intervertebral disc fusion will cause stress concentration in the annulus fibrosus or adjacent vertebral bones, leading to accelerated degeneration of the damaged intervertebral disc, recurrence of the nucleus pulposus or herniation of adjacent intervertebral discs. How to restore the biological function of the damaged intervertebral disc remains a difficult problem in clinical practice.

[0003] Due to the excellent hydration ability similar to that of the nucleus pulposus tissue, hyaluronic acid-based hydrogels have been widely used in the treatment of degenerative intervertebral discs. However, unfortunately, although a variety of hyaluronic acid-based hydrogels have been constructed, most of them are not suitable for nucleus pulposus regeneration. For example, the ultraviolet light-crosslinked hydrogel prepared from methacryloyl hyaluronic acid (HAMA) is fragile and difficult to inject through a syringe; aldehyde group-modified hyaluronic acid (HA-ADH) can form a hydrogel through Schiff base reaction and shows good adhesion to tissues, but excessive aldehyde groups may induce cytotoxicity and biological side effects. In addition, hyaluronic acid physical gels can also be prepared by freeze-thaw cycles in an acidic environment, but due to the harsh preparation conditions, the application of hyaluronic acid frozen physical gels in cell delivery is limited.

[0004] Currently, the research on cell therapy using hyaluronic acid solution loaded with mesenchymal stem cells for the treatment of degenerative intervertebral discs has entered the clinical research stage. However, the anoikis apoptosis of implanted exogenous stem cells and low cell survival rate pose major challenges to the clinical treatment efficacy of degenerative intervertebral discs. In the early stage of intervertebral disc degeneration, harsh microenvironmental factors in the intervertebral disc, such as inflammation, hypoxia and acidic environment, can further induce cell damage, such as programmed cell death, exacerbation of inflammation and nerve ingrowth, etc., further aggravating the degeneration process of the intervertebral disc. Moreover, the liquid state of the hyaluronic acid solution carrier will cause inevitable cell leakage, resulting in the formation of osteophytes.

[0005] Therefore, for the treatment of degenerative intervertebral discs, it is crucial to design a cell delivery system that can regulate the microenvironment of the degenerated site and ensure the viability of supplemented exogenous cells. And, in order to ensure that the encapsulated cells can accurately remain at the implanted site, the delivery system also needs to have appropriate viscoelasticity and adhesiveness. Summary of the Invention

[0006] The present invention overcomes the deficiencies in the prior art. For example, existing intervertebral disc regeneration scaffold materials have poor hydration ability, low biological activity, and are difficult to adapt to the degenerative intervertebral disc microenvironment. As a result, it is difficult to ensure the activity of exogenous cells and restore the biomechanical function of the intervertebral disc. The present invention provides a self-gelling hyaluronic acid-based physically crosslinked hydrogel, its preparation method and application. The preparation method of the present invention is simple. By grafting thymine onto hyaluronic acid, a physically crosslinked hyaluronic acid hydrogel driven by hydrogen bonds is achieved. Physical crosslinking retains the excellent hydration ability of hyaluronic acid and endows the hydrogel with injectability and adhesiveness. The doping of inorganic nanoparticles manganese dioxide improves the adaptability of the hydrogel in 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. The application potential of the present invention in intervertebral disc regeneration is preliminarily confirmed through animal experiments.

[0007] The object of the present invention is achieved by the following technical solutions.

[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, successively add carbohydrazide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 1-hydroxybenzotriazole (HOBT). After reacting for 24 - 96 h, transfer the reaction solution to a dialysis bag, and dialyze and freeze-dry to obtain hydrazide-modified hyaluronic acid (HA-HYD);

[0010] Among them, the molecular weight of hyaluronic acid is 20,000 - 100,000, the concentration of hyaluronic acid in the reaction system is 5‰ - 15‰, and the molar ratio of carbohydrazide, 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 hydrazide-modified hyaluronic acid (HA-HYD) prepared in Step 1 in ultrapure water. After it is completely dissolved, successively add thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS). Adjust the pH of the above reaction system to 4 - 7. After reacting for 12 - 72 h, transfer the reaction solution to a dialysis bag, and dialyze and freeze-dry to obtain thymine-grafted hyaluronic acid (HAT);

[0012] Among them, 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 and homogenize it, and then let it stand at room temperature to obtain thymine-hyaluronic acid hydrogel (HAT), that is, a self-gelling hyaluronic acid-based physically cross-linked hydrogel;

[0014] Among them, 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 carbohydrazide, 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 48 h.

[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 h, 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 a physical gel driven by hydrogen bonds without a cross-linking agent; the storage modulus of the gel is 40 - 2000 Pa; the hydration capacity of the 5% thymine-hyaluronic acid (HAT) hydrogel is 20.79 ± 2.76, 17.28 ± 1.36, 7.07 ± 0.29, 4.73 ± 0.07, 3.78 ± 0.31 g H 2 O / g at the osmotic pressures of 0.3, 1.1, 2.5, 4.4, and 6.9 atm, and the adhesion strength is 1 - 3 kPa.

[0019] Self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold: The self-gelling hyaluronic acid-based physically cross-linked hydrogel prepared above is combined with bone marrow mesenchymal stem cells to form a self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold (HAT-MSCs).

[0020] A manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel and its preparation method. In step 3, the thymine-modified hyaluronic acid prepared in step 2 is dissolved in a PBS buffer solution dispersed with manganese dioxide, vortexed and homogenized, and after standing at room temperature, a manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn) is obtained, that is, a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel;

[0021] Among them, in the PBS buffer solution dispersed with manganese dioxide, the concentration of manganese dioxide is 100 μg / ml - 500 μg / ml.

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

[0023] A manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold is formed by doping the above-prepared manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel with bone marrow mesenchymal stem cells to form a manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold (HATMn-MSCs).

[0024] The application of the self-gelling hyaluronic acid-based physically crosslinked hydrogel, the self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold, the manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel or the manganese dioxide-doped self-gelling hyaluronic acid-based physically crosslinked hydrogel scaffold in biomedical materials, especially for medical materials used in intervertebral disc regeneration.

[0025] The beneficial effects of the present invention are as follows: The preparation method of the present invention is simple. By grafting thymine on hyaluronic acid, a hydrogen bond-driven self-gelling hyaluronic acid physical gel is realized. Physical crosslinking retains the hydration ability of the gel and endows the gel with injectability and adhesiveness. The doping of inorganic nanoparticles manganese dioxide is used to improve the adaptability of the gel in 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. Animal experiments preliminarily confirm its application potential in intervertebral disc regeneration. Description of the Drawings

[0026] Figure 1 is the NMR spectrum of hydrazide-modified hyaluronic acid, thymine-1-acetic acid and thymine-hyaluronic acid (HAT);

[0027] Figure 2 is the Fourier transform infrared spectrum of hydrazide-modified hyaluronic acid, thymine-1-acetic acid and thymine-hyaluronic acid (HAT);

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

[0029] Figure 4 It is the adhesion property characterization of thymine-hyaluronic acid in the present invention. Among them, (a) is the adhesion schematic diagram of HAT gel to iron, ceramic, wood, glass, plastic, polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET); (b) is the adhesion schematic diagram of HAT gel to small intestine, bone, skin, heart, and liver; (c) is the lap shear adhesion force characterization diagram of HAT gel to pig skin.

[0030] Figure 5 It is the performance characterization of HAT doped with MnO 2 nanoparticles for HATMn to scavenge ROS. Among them, (a) is the statistical chart of the efficiency of HATMn to scavenge hydrogen peroxide; (b) is the statistical chart of the efficiency of HATMn to scavenge hydroxyl radicals; (c) is the live-dead staining of MSCs under the culture medium control group (control), hydrogen peroxide treatment (H 2 O 2 ), treatment with both HATMn and hydrogen peroxide (HATMn + H 2 O 2 ).

[0031] Figure 6 It is the results of hematoxylin-eosin staining (a) and alcian blue staining (b) at different time points of using HATMn-MSCs for the treatment of rat caudal intervertebral disc degeneration in the present invention. Detailed implementation mode

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

[0033] Example 1

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

[0035] Step 1: First, weigh 1 g of HA (Mw = 50,000) into a 250 mL round-bottom flask, add 150 mL of ultrapure water as a solvent, stir to completely dissolve HA, then add 0.2 g of carbohydrazide, stir to completely dissolve it, then add 0.5 g of 1-hydroxybenzotriazole (HOBT) and 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), adjust the pH to 4, and react at 25 °C for 24 h. After the reaction is completed, dialyze in 0.1 M 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, stir to completely dissolve HA-HYD, then add 0.5 g of thymine-1-acetic acid, stir to completely dissolve it, then 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 completed, dialyze in 0.1 M NaCl solution for two days, dialyze in pure water for one day, and freeze-dry to obtain thymine-hyaluronic acid (HAT).

[0037] Step 3: Weigh 0.05 g of HAT using an analytical balance and place it in a centrifuge tube. Add 1 mL 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 h to obtain thymine-hyaluronic acid hydrogel (HAT), that is, a self-gelling hyaluronic acid-based physical 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 = 50,000) into a 250 mL round-bottom flask, add 150 mL of ultrapure water as a solvent, stir to completely dissolve HA, then add 0.54 g of carbohydrazide, stir to completely dissolve it, then 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 completed, dialyze in 0.1 M NaCl solution for two days, dialyze in 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 1 g of HA-HYD into a 250 mL round-bottom flask, add 100 mL of ultrapure water as a solvent and stir to completely dissolve HA-HYD. Then add 0.6 g of thymine-1-acetic acid and stir to completely dissolve it. 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 24 h. After the reaction is completed, 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).

[0042] Step 3: Weigh 0.1 g of HAT using an analytical balance and place it in a centrifuge tube. Add 1 ml 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 h to obtain thymine-hyaluronic acid hydrogel (HAT), which is a self-gelling hyaluronic acid-based physically crosslinked hydrogel.

[0043] Example 3

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

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

[0046] Step 2: Preparation of thymine-hyaluronic acid (HAT): First, weigh 2 g of HA-HYD into a 250 mL round-bottom flask, add 100 mL of ultrapure water as a solvent and stir to completely dissolve HA-HYD. Then add 1 g of thymine-1-acetic acid and stir to completely dissolve it. 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 48 h. After the reaction is completed, 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).

[0047] Step 3: Weigh 0.15 g of HAT using an analytical balance and place it in a centrifuge tube. Add 1 ml 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 h to obtain thymine-hyaluronic acid hydrogel (HAT), that is, self-gelling hyaluronic acid-based physically cross-linked hydrogel.

[0048] Example 4:

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

[0050] Example 5:

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

[0052] Example 6:

[0053] Weigh 5 mg of manganese dioxide (MnO 2 ) and place it in a 15-ml centrifuge tube. Add 10 ml of PBS and sonicate to disperse manganese dioxide (MnO 2 ) evenly; weigh 0.15 g of HAT prepared in Step 2 of Example 3 using an analytical balance, place it in a centrifuge tube, add 1 ml of PBS in which manganese dioxide (MnO 2) of PBS, the vortex makes HAT uniformly mixed in PBS dispersed with manganese dioxide, and then it is left standing at room temperature for 2 h to obtain manganese dioxide-doped thymine-hyaluronic acid hydrogel (HATMn), that is, manganese dioxide-doped self-gel hyaluronic acid-based physical cross-linked hydrogel.

[0054] Nuclear magnetic resonance and infrared characterization of HAT gel: As Figure 1 shown, in the 1 H NMR spectrum of thymine-1-acetic acid, the signals at 1.86, 4.51, and 7.4 ppm are clearly shown, and these signals correspond to the hydrogens of the methyl (a), methylene (b), and alkene (c) in thymine-1-acetic acid, respectively. By comparing the NMR spectra of HA and HAT, the successful synthesis of HAT was proved. In the HAT spectrum, in addition to showing the sugar ring structure in HA 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. As Figure 2 shown, FTIR analysis clarified the hydrogen bond-driven gelation mechanism of HAT gel. Compared with HA, the symmetric stretching vibration corresponding to C=O and the vibration peak of amide II band near 1635 cm -1 and 1560 cm -1 are slightly red-shifted in HAT, indicating the formation of hydrogen bonds between C=O groups.

[0055] Test on the hydration ability and swelling behavior of HAT gel: First, solutions with different osmotic pressures (0.03 - 0.69 MPa) are prepared using PEG (MW = 20,000). Then, record the mass of the freeze-dried HAT as W 0 , put it into a dialysis tube (8,000 MWCO), and then immerse the dialysis tube in PEG solutions with different osmotic pressures for 24 hours, and record the mass of the hydrated gel again, denoted as W 1 . The hydration ability of HAT is calculated by the following formula:

[0056]

[0057] The swelling behavior of the gel is determined by soaking the freeze-dried gel in a PBS environment, and at 37 °C, the weight of the gel is weighed again at regular intervals, and it is calculated by the increased mass ratio. As Figure 3 shown, HAT gel has good hydration ability, and the hydration abilities at 0.3 atm, 1.1 atm, 2.5 atm, 4.4 atm, and 6.9 atm are 20.79 ± 2.76, 17.28 ± 1.36, 7.07 ± 0.29, 4.73 ± 0.07, 3.78 ± 0.31 g H 2O / g. Moreover, the swelling results show that 5% HAT can swell up to 22 times its weight, and 15% HAT can swell up to 45 times its original weight. The phenomenon that the swelling degree of the gel increases with the increase in solid content can be attributed to the fact that as the solid content of the gel increases, the structural stability of the gel becomes stronger, and it takes longer for the structure to collapse (the weight of the hydrogel of 5% HAT has decreased at 58.5 hours, that of 10% HAT at 84 hours, and that of 15% HAT at 116 hours), resulting in a higher swelling rate in PBS.

[0058] Rheological property test of HAT gel: The gel state of HAT was determined using the time sweep mode. As Figure 3 shown, the storage modulus of the HAT gel is greater than the loss modulus, indicating that HAT is in the gel state. While HA-HYD all show solution behavior with the loss modulus greater than the storage modulus. The variable strain sweep rheological spectrum under the oscillation mode shows that HAT has good shear thinning behavior and can be smoothly extruded from the syringe. Due to the presence of a large number of hydrogen bonds in the HAT gel, HAT also exhibits good self-healing performance. When two gels are gently placed together, they heal into a whole after 1 minute and can withstand deformations such as stretching and torsion.

[0059] Characterization of the adhesion behavior of HAT gel: First, 10% HAT gel was prepared, and then the interaction between the HAT gel and different materials was attempted to investigate the adhesion between the gel and the materials. As Figure 4 shown, the HAT gel has adhesion 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 small intestine, bone, skin, heart, and liver. Using pig skin as the adhesion model, the adhesion strength of the gel to pig skin was quantitatively characterized by lap shear test. It can be seen from the test results that as the solid content of the gel increases, the adhesion strength also gradually increases. The adhesion strength of the gel with a solid content of 15% to pig skin can reach 3.5 kPa. Moreover, the adhesion failure of the gel is mainly caused by the damage of the bulk.

[0060] H of HATMn gel 2 O 2 and OH· capture capacity test: MnO 2 nanoparticles were doped in the HAT gel at concentrations of 200 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml, and 1000 μg / ml, and the capture capacity of HATMn for H 2 O 2 and OH· was determined. First, a Ti(SO4) 2 solution was prepared, and 0.2 M H 2 O2 After co-incubation with HATMn for 10 min, 2.6 ml of Ti(SO4) 2 solution was added to detect the residual H 2 O 2 . Then, the absorbance of the supernatant at 405 nm was measured using a microplate reader. For the determination of OH· scavenging ability, 9 mM FeSO 4 , 8.8 mM H 2 O 2 and 9 mM salicylic acid solution were first prepared, and then 200 μl of 9 mM FeSO 4 and 200 μl of 8.8 mM H 2 O 2 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. As Figure 5 shown, the ability of HATMn to capture free radicals increased with the increase of MnO 2 concentration. When the MnO 2 concentration was 200 μg / ml, the HATMn's ability to capture H 2 O 2 reached 70%, while when the MnO 2 concentration was 400 μg / ml, the HATMn's ability to capture H 2 O 2 almost reached 100%. When the MnO 2 concentration was 1000 μg / ml, the HATMn's ability to capture OH· reached 70%.

[0061] Test on the ability of HATMn gel to protect cells from H 2 O 2 damage: First, mesenchymal stem cells were seeded at 1×10 5 cells / well in a 12-well plate. After culturing for 24 h, the medium was replaced with a conditioned medium containing 400 μM of H 2 O 2 , and HATMn gel was added to the well plate. After culturing for 4 h, the cells were stained with a live / dead cell stain (Calcein-AM / PI), and finally photographed using an inverted fluorescence microscope. As Figure 6 shown, in the group without HATMn gel, many cells showed PI positivity, indicating that the cell state was damaged, while in the HATMn group, almost all cells showed AM positivity and PI negativity, indicating that the cell state was good, proving that HATMn could effectively protect cells from H 2 O 2 damage.

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

[0063] Evaluation of the therapeutic effect of the HATMn-MSCs scaffold on rat caudal intervertebral disc degeneration: First, a rat caudal intervertebral disc degeneration model was established and waited for 4 weeks to ensure the successful establishment of the model. At the 5th week, HA-MSCs or HATMn-MSCs were injected into the nucleus pulposus site using a microsyringe, and the corresponding intervertebral discs were named the HA-MSCs group and the HATMn-MSCs group, respectively. As a control, the punctured intervertebral disc and the normal intervertebral disc were also set up and named the IVDD group and the normal group, respectively. At the 4th, 8th, and 12th weeks after the administration of the HA-MSCs and HATMn-MSCs materials, samples were taken for fixation, decalcification, hematoxylin-eosin staining, and Alcian blue staining. As Figure 6 shown in a, the hematoxylin-eosin (HE) staining images showed that compared with the normal IVD, the content of the nucleus pulposus tissue in the IVDD group gradually decreased over time, and at 12 weeks, the boundary between the nucleus pulposus and the annulus fibrosus in the IVD was blurred. The NP in the HA-MSCs group continued to degenerate, resulting in the rupture of the AF and invasion into the NP space. Therefore, the residual amount of NP was small at 12 weeks. In contrast, in the HATMn-MSCs group, a clear boundary between the NP and the AF was observed at each time point. The fibrous tissue in the AF was arranged regularly. Different from the aggregation of inflammatory cells in the IVD region in the IVDD group and the HA-MSCs group, the HATMn-MSCs group showed a smaller inflammatory response. As Figure 6 shown in b, the Alcian blue staining showed that the IVD in the normal group showed obvious proteoglycan staining in the NP region, while showing good collagen arrangement and very few cells in the AF region. The Alcian blue staining in the IVDD group was not obvious. After treatment with HA-MSCs, the proteoglycan staining outside the AF and the heterogeneity of the NP still increased. In contrast, the distribution of proteoglycans and collagen in the intervertebral disc in the HATMn-MSCs group was more regular. In addition, we also observed that the collagen was arranged neatly, showing a typical natural morphology, similar to the natural intervertebral disc. To sum up, the above results further verified the effectiveness of the HATMn-MSCs strategy in delaying the progression of IVDD and promoting NP regeneration.

[0064] The above has made an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification, or equivalent replacement that can be made by those skilled in the art without creative labor falls within the protection scope of the present invention.

Claims

1. A method for preparing a self-gelling hyaluronic acid-based physically cross-linked hydrogel, characterized in that: Follow the steps below: Step 1, dissolving sodium hyaluronate powder in ultrapure water, and after it is completely dissolved, sequentially adding dihydrazide carbonate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole, reacting for 24-96 hours, transferring the reaction solution to a dialysis bag, dialyzing and freeze-drying to obtain hydrazide hyaluronic acid; 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 carbonate dihydrazide, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is (1-5): (1-3): 1: 2; Step 2, dissolving the hydrazide hyaluronic acid prepared in step 1 in ultrapure water, and after it is completely dissolved, sequentially adding thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide, and adjusting the pH of the reaction system to 4-7, reacting for 12-72 hours, transferring the reaction solution to a dialysis bag, dialyzing and freeze-drying to obtain thymine-grafted hyaluronic acid; The concentration of hydrazide hyaluronic acid in the reaction system is 5‰-2%, and the mass ratio of hydrazide hyaluronic acid, thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and N-hydroxysuccinimide is (1-5): (1-3): 1: 1; Step 3, dissolving the thymine-modified hyaluronic acid prepared in step 2 in PBS buffer, vortexing and homogenizing, and standing at room temperature to obtain a thymine-hyaluronic acid hydrogel, i.e., a self-gelling hyaluronic acid-based physically cross-linked hydrogel; The concentration of thymine-hyaluronic acid is 1-20%.

2. The method for preparing a self-gelling hyaluronic acid-based physically cross-linked hydrogel according to claim 1, characterized in that: In step 1, the molecular weight of hyaluronic acid is 5w, the concentration of hyaluronic acid in the reaction system is 8‰, the molar ratio of carbonate dihydrazide, sodium hyaluronate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 2:1:1:2, and the reaction is carried out at 25°C for 48h; In step 2, the concentration of hydrazide 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 hyaluronic acid, thymine-1-acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 2:1:0.1:0.1; In step 3, the concentration of thymidine-hyaluronic acid is 5%.

3. A self-gelling hyaluronic acid-based physically cross-linked 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: The self-gelling hyaluronic acid-based physically cross-linked hydrogel as claimed in claim 3 is combined with bone marrow mesenchymal stem cells to form a self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold.

5. Use of the self-gelling hyaluronic acid-based physically cross-linked hydrogel as claimed in claim 3 or the self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold as claimed in claim 4 in biomedical materials, especially medical materials for intervertebral disc regeneration.

6. A method for preparing a manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel based on the preparation method of claim 1 or 2, characterized in that: In step 3, the thymine-modified hyaluronic acid prepared in step 2 is dissolved in a PBS buffer solution dispersed with manganese dioxide, vortexed and homogenized, and allowed to stand at room temperature to obtain a manganese dioxide-doped thymine-hyaluronic acid hydrogel, i.e., a manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel; The concentration of manganese dioxide in the PBS buffer in which manganese dioxide is dispersed is 100 μg / ml-500 μg / ml.

7. The method for preparing a manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel according to claim 6, characterized in that: In step 3, the concentration of manganese dioxide in the PBS buffer in which manganese dioxide is dispersed is 200 μg / ml.

8. A manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel prepared by the preparation method according to claim 6 or 7.

9. A manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold, characterized in that: The manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel as claimed in claim 8 is combined with bone marrow mesenchymal stem cells to form a manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold.

10. Use of the manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel as claimed in claim 8 or the manganese dioxide-doped self-gelling hyaluronic acid-based physically cross-linked hydrogel scaffold as claimed in claim 9 in biomedical materials, especially medical materials for intervertebral disc regeneration.

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