CN117462749A - Environment-adaptive dynamic hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention discloses an environment-adaptive dynamic hydrogel, which is characterized by comprising OHA, PVA and sodium tetraborate; NMO or GDF-5. The present invention can produce self-healing HPGO containing more dynamic bonds by injecting hydrogel precursors including OHA-GDF5, NMO and PVA into the intervertebral disc under the action of sodium tetraborate, and can slowly release GDF-5 and NMO as a local drug library while providing mechanical support for the intervertebral disc. In addition, as the rich Schiff base groups of HPGO form a molecular network capable of adsorbing free ferrous iron molecules, excessive free ferrous iron is one of physiological characteristics of disc degeneration, and after the HPGO adsorbs excessive ferrous ions of the disc, the crosslinking level of HPGO can be obviously improved, the mechanical elasticity of the HPGO is enhanced, and the resistance of the HPGO to compression load in IVD is improved. The hydrogel can release the medicine; the treatment effect is good; finally, the self-healing effect is achieved on the supporting capability.

Description

Environment-adaptive dynamic hydrogel and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to an environment-adaptive dynamic hydrogel, and a preparation method and application thereof.

Background

Lumbago is the most common bone and muscle diseases in modern society, wherein intervertebral disc degeneration is one of the main causes of lumbago, is a chronic disease related to age, often causes disorder of crowd activities and loss of labor force, brings great burden to social economy and medical care, and still lacks effective treatment means for reversing the intervertebral disc degeneration disease course clinically at present. The nucleus pulposus tissue is positioned in the center of the intervertebral disc, is vital to maintain the physiological and mechanical functions of the intervertebral disc, and is an ideal method for treating the degeneration of the intervertebral disc. Therefore, how to maintain the mechanical function of the disc and at the same time restore the physiological function of the nucleus pulposus tissue is one of the primary problems in the treatment of disc degeneration.

The hydrogel is a novel material in the field of biological medicine, has good biocompatibility, plasticity, degradability and the like, can carry and release medicines, and is one of ideal materials for repairing damaged tissues. However, the hydrogel is usually fixed in structure after crosslinking, which is often far from various complex biological structures in the body, resulting in a decrease in therapeutic effect. The environment-adaptive dynamic hydrogel is a novel intelligent hydrogel suitable for tissue regeneration, has the characteristic of reversible crosslinking, can drive the inside of the hydrogel to generate physical or chemical change through the tissue microenvironment, and release functional factors or change the crosslinking degree of the hydrogel, so that the performance of the hydrogel is subjected to environment-adaptive reprogramming, and finally, a better tissue reconstruction effect is achieved.

Hydrogels are networks of hydrophilic polymer chains, sometimes also called colloidal gels, in which water is the dispersing medium. Three-dimensional software is due to the hydrophilic polymer chains being held together by physical or chemical cross-linking. Due to the inherent cross-linking, the structural integrity of the hydrogel network is not dissolved by high concentrations of water. Conventional hydrogels are crosslinked by various covalent bonds and are irreversible, so that the microstructure is usually fixed, often destroyed in a complex in vivo environment and difficult to recover, which is detrimental to mechanical support of the tissue. And the cells will interact with the surrounding environment through a combination of static and dynamic mechanical signals, the poor extracellular matrix environment will also limit their growth and function, and thus the therapeutic effect of the non-dynamic hydrogel structure is limited.

The dynamic hydrogel is hydrogel containing multiple dynamic bonds (acylhydrazone bonds, imine bonds, hydrogen bonds and the like), the dynamic bonds have excellent self-healing performance, the dynamic bonds can be filled into the intervertebral disc target part in an injection mode, then the dynamic bonds contained in the hydrogel are quickly recombined, the internal gel-like structure is rebuilt, and mechanical support is provided for subsequent tissue repair. In addition, the method can reprogram the internal network structure of the hydrogel and release the medicine according to the acidity of the degenerated intervertebral disc microenvironment and the characteristic of harmful ferrous ions, thereby enhancing the lasting treatment effect on the structure and the medicine, further regulating the related cell behaviors and providing proper conditions for the repair of the intervertebral disc tissues.

The mechanical and physical and chemical environments in the nucleus pulposus are complex, the non-dynamic hydrogel is often damaged and the treatment effect is reduced, the dynamic hydrogel can self-heal, the microenvironment and the cell behavior of the intervertebral disc are improved from the two aspects of mechanics and drug release, and the tissue reconstruction of the intervertebral disc is promoted so as to treat the degeneration of the intervertebral disc.

Therefore, the invention prepares the environment self-adaptive dynamic hydrogel with the performance regulated by the components of the microenvironment of the intervertebral disc, which is used for improving the microenvironment of the nucleus pulposus and delaying the degeneration process of the intervertebral disc.

Disclosure of Invention

The invention firstly provides an environment-adaptive dynamic hydrogel, which comprises OHA, PVA and sodium tetraborate; NMO or GDF-5.

Preferably, the NMO preparation method includes: extracting nucleus pulposus cell membrane (nano vesicle NM) by repeated extrusion by adopting a liposome extrusion method, mixing the extracted NM with Ori in a volume ratio of 2:1 in a liposome extruder, and forming NMO by repeated extrusion.

Preferably, the method for synthesizing Oxidized Hyaluronic Acid (OHA) comprises the steps of: 10mg of Hyaluronic Acid (HA) was dissolved in 100mL of distilled water, followed by the addition of 20mM sodium periodate; the mixture was stirred for 24 hours in the dark; thereafter, the mixture was dialyzed in the dark (dialysis bag MW 3500 Da) for 3 days and dried under vacuum to obtain aldehyde group-containing Oxidized Hyaluronic Acid (OHA) for the subsequent experiments.

Preferably, the OHA concentration is 5-15mg/mL, the PVA concentration is 5-15% w/v, and the sodium tetraborate concentration is 0.5-1.5% w/v.

Preferably, the OHA concentration is 10mg/mL, the PVA concentration is 10% w/v, and the sodium tetraborate concentration is 1% w/v.

The invention also provides a preparation method of the environment-adaptive dynamic hydrogel, which comprises the following steps: (1) Preparation and detection of Ori-loaded Nanovesicles (NMO); (2) Preparing and detecting hydrogel precursors, namely Oxidized Hyaluronic Acid (OHA); (3) preparation and detection of dynamic hydrogel.

Preferably, the preparation of the NMO includes: extracting nucleus pulposus cell membrane (nano vesicle NM) by repeated extrusion by adopting a liposome extrusion method, mixing the extracted NM with Ori in a volume ratio of 2:1 in a liposome extruder, and forming NMO by repeated extrusion.

Preferably, the preparation of Oxidized Hyaluronic Acid (OHA) comprises: 10mg of Hyaluronic Acid (HA) was dissolved in 100mL of distilled water, followed by the addition of 20mM sodium periodate; the mixture was stirred for 24 hours in the dark; thereafter, the mixture was dialyzed in the dark (dialysis bag MW 3500 Da) for 3 days and dried under vacuum to obtain aldehyde group-containing Oxidized Hyaluronic Acid (OHA) for the subsequent experiments.

Preferably, the preparation of the dynamic hydrogel comprises: 10mg of OHA was dissolved in 100mL of PBS (pH=7.4) to obtain 10mg/mL of OHA solution; meanwhile, 1g of polyvinyl alcohol (PVA) was dissolved in 10mL of PBS (ph=7.4) at 90 ℃ to obtain a 10% PVA solution; mixing 300. Mu.L 10% PVA solution and 700. Mu.L OHA solution (10 mg/mL) at room temperature; the mixture was then added to 1% sodium tetraborate to immediately initiate the formation of a composite hydrogel, designated HP, at room temperature; similarly, GDF5, NMO and GDF5+NMO were added to a mixture of 300. Mu.L 10% PVA solution and 700. Mu.L OHA solution (10 mg/mL) to form HPG, HPO and HPGO hydrogels, respectively, after the introduction of 1% sodium tetraborate.

The invention also provides an environment-adaptive dynamic hydrogel or an application of the method for preparing the environment-adaptive dynamic hydrogel, wherein the application is a) preparation of a bioengineering reagent or a kit; or b) preparing a reagent or kit for repairing or reconstructing the tissue of the intervertebral disc; or c) preparing a medicament for treating intervertebral discs; or d) a method of restoring the function of a nucleus pulposus cell, optionally for non-diagnostic or non-therapeutic purposes.

Compared with the prior art, the invention has at least the following beneficial effects:

the Ori-loaded Nanovesicle (NMO) has the beneficial effects that: oridonin (Ori) is a natural terpenoid found in traditional Chinese herbal medicines, has a powerful anti-inflammatory effect, and has led to great interest in IDD treatment. However, its inherent hydrophobicity severely limits clinical applications. To address this challenge, we extracted the nucleus pulposus cell membrane (NM) by repeated extrusion using liposome extrusion and used it to prepare Ori-loaded nanovesicles, which would be expected to promote Ori incorporation while enabling its homologous delivery to nucleus pulposus cells in an inflammatory environment.

The beneficial effects of hydrogel precursors, namely Oxidized Hyaluronic Acid (OHA) (i.e. the beneficial effects of hydrogel scaffold): most current dynamic hydrogel designs have low mechanical resilience and often lead to premature hydrogel fracture and insufficient IVD regeneration after being subjected to excessive mechanical stress. The invention designs hydrogel based on PVA and an OHA as a framework, wherein the OHA and the PVA can establish various dynamic bonds to provide mechanical support for a degenerated intervertebral disc, and the hydrogel can be prevented from being broken prematurely.

The hydrogel has the beneficial effects that: injection of hydrogel precursors including OHA-GDF5, NMO and PVA into the disc under the action of sodium tetraborate produces self-healing HPGO with more dynamic bonds, which slowly releases GDF-5 and NMO as a local depot while providing mechanical support to the disc. In addition, as the rich Schiff base groups of HPGO form a molecular network capable of adsorbing free ferrous iron molecules, excessive free ferrous iron is one of physiological characteristics of disc degeneration, and after the HPGO adsorbs excessive ferrous ions of the disc, the crosslinking level of HPGO can be obviously improved, the mechanical elasticity of the HPGO is enhanced, and the resistance of the HPGO to compression load in IVD is improved. The hydrogel can release the medicine; the treatment effect is good; finally, the self-healing effect is achieved on the supporting capability.

Drawings

FIG. 1 is a transmission electron micrograph of nuclear membrane NM and Ori-loaded Nanovesicles (NMO);

fourier transform infrared spectroscopy analysis (left) for OHA, nuclear magnetic resonance hydrogen spectrogram (right) of fig. 2;

FIG. 3 primary crosslinking effect;

FIG. 4 is a schematic diagram of a dynamic hydrogel after primary crosslinking and a scanning electron microscope after secondary crosslinking under the action of ferrous ions;

FIG. 5 identification of dynamic hydrogels binding to ferrous ions;

FIG. 6 drug release curves of hydrogels after primary crosslinking and after secondary crosslinking under different conditions;

FIG. 7 biocompatibility testing of dynamic hydrogels; the left figure is a schematic representation of co-culture of hydrogel and nucleus pulposus cells, the hydrogel being immersed in a culture medium together with the cells through a scaffold. The right panel shows the results of cell activity assays for co-culture, with no significant difference in NPC activity for dynamic hydrogel co-culture versus control without hydrogel co-culture (p > 0.05);

FIG. 8 important mechanical functions; the upper diagram is a schematic diagram, the left is a schematic diagram of a measurement mode, the middle is the crosslinking effect of two dynamic hydrogels, and the right is a schematic diagram thereof

The lower graph shows the measurement results, the left graph shows the primary crosslinking and the reaction between Fe ²⁺ Compression performance results after ion-induced secondary crosslinking; the right figure shows the compression of the basic hydrogel HP and the 2nd-HP after secondary crosslinking and the non-dynamic hydrogel N-HP and the non-dynamic hydrogel N-HPGO loaded with drugsPerformance results;

FIG. 9 therapeutic effect 1 cell viability assay after 48h co-culture of nucleus pulposus cells with hydrogel under inflammation induction; the left panel shows co-culture of four hydrogels with nucleus pulposus cells comprised by the present invention; the right graph shows the basic hydrogel HP, the hydrogel HPGO with the best treatment effect and the non-dynamic hydrogel N-HP, wherein the pure drug GO (100 ng/ml) is used, and the non-dynamic hydrogel carries the co-culture of the drug N-HPGO and the nucleus pulposus cells;

FIG. 10 treatment effects 2 treatment effects of different hydrogels after rat tail disc penetration; the upper graph shows the therapeutic effect of four hydrogels encompassed by the present invention in a rat tail disc puncture model; the lower graph shows the therapeutic effects of the basic hydrogel HP, the hydrogel HPGO with the best therapeutic effect and the non-dynamic hydrogel N-HP, the pure administration GO and the non-dynamic hydrogel carried drug N-HPGO in a rat model.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Hyaluronic acid, polyvinyl alcohol, sodium tetraborate and sodium periodate were purchased from aladin limited (china). Oridonin (HY-N0004) and GDF5 (HY-P72633) were purchased from MedChemexpress (Shanghai, china). Tert-butyl hydroperoxide (TBHP) solution was purchased from Sigma (458139). Other reagents were purchased from Chuandong chemical company (Chongqing, china).

Example 1 preparation and detection of Ori-loaded Nanovesicles (NMO)

Extracting nucleus pulposus cell membrane (nanometer vesicle NM) by repeated extrusion by adopting a liposome extrusion method, mixing the extracted NM with Ori in a volume ratio of 2:1 in a liposome extruder, and forming NMO by repeated extrusion.

When the cell number reaches 3×10 ⁷ At each time, NPC was digested with 0.25% trypsin and centrifuged at 5000rpm for 15 minutes to obtain a cell pellet. The cell pellet was then resuspended in PBS and freeze-thawed 3 times, and then centrifuged at 10000rpm for 15min to give a supernatant containing NPC membrane. 1mL of the supernatant was repeatedly extruded with a liposome extruder for 35 cycles to obtain NPC film, which was combined with 0.5mL of oridonin (Ori) (4μg/mL) was further mixed. Then, 1.5mL of the mixture was repeatedly extruded with a liposome extruder for 35 cycles to achieve uniform coating of NPC film on Ori, thus obtaining Ori-loaded Nanovesicles (NMO).

Example 2 detection: characterization of morphology and drug loading by Transmission Electron microscopy

Transmission Electron Microscopy (TEM) imaging was performed on a JEOL JEM-2010TEM (200 kV acceleration voltage) to observe the morphology of NPC films (NMM) and NMO. The results (fig. 1) demonstrate that both NM and NMO have excellent dispersibility and maintain a uniform spherical morphology. The average size of NMO is slightly increased compared to NM due to the inclusion of hydrophobic Ori drugs in the core compartment.

EXAMPLE 3 preparation and detection of hydrogel precursor, oxidized Hyaluronic Acid (OHA)

Synthesis of Oxidized Hyaluronic Acid (OHA): 10mg of Hyaluronic Acid (HA) was dissolved in 100mL of distilled water, followed by the addition of 20mM sodium periodate. The mixture was stirred for 24 hours in the dark. Thereafter, the mixture was dialyzed in the dark (dialysis bag MW 3500 Da) for 3 days and dried under vacuum to obtain aldehyde group-containing Oxidized Hyaluronic Acid (OHA) for the subsequent experiments.

Example 4 detection: fourier transform infrared spectrum analysis and nuclear magnetic resonance hydrogen spectrogram

OHA containing aldehyde groups is obtained by an oxidation reaction between Hyaluronic Acid (HA) and sodium periodate. FTIR spectroscopic analysis (left in FIG. 2) showed that the characteristic absorption peak of aldehyde group appeared at 1722cm ^-1 Successful introduction of aldehyde groups on the OHA chain was confirmed. Meanwhile, the 1H NMR results (right in FIG. 2) showed that a new chemical shift of aldehyde groups in OHA samples occurred at 4.9-5.0ppm, indicating that aldehyde groups on OHA formed hemiacetal protons with adjacent hydroxyl groups, again confirming successful preparation of aldehyde-containing OHA.

Example 5 preparation and detection of dynamic hydrogels

By mixing 10mg/mL of OHA, 10% w/v PVA and 1% w/v sodium tetraborate, crosslinking at room temperature and adding 5. Mu.g NMO and/or 5. Mu.g GDF-5 as required during the process, dynamic hydrogels HPGO, HPG, HPO loaded with NMO and/or GDF-5 were finally obtained.

10mg of OHA was dissolved in 100mL of PBS (pH=7.4) to obtain 10mg/mL of OHA solution. Meanwhile, 1g of polyvinyl alcohol (PVA) was dissolved in 10mL of PBS (ph=7.4) at 90 ℃ to obtain a 10% PVA solution. mu.L of 10% PVA solution and 700. Mu.L of OHA solution (10 mg/mL) were stirred and mixed at room temperature. The mixture was then added to 1% sodium tetraborate to immediately initiate the formation of a composite hydrogel, designated HP, at room temperature. Similarly, GDF5, NMO and GDF5+NMO were added to a mixture of 300. Mu.L 10% PVA solution and 700. Mu.L OHA solution (10 mg/mL) to form HPG, HPO and HPGO hydrogels, respectively, after the introduction of 1% sodium tetraborate.

Preparation of the non-dynamic hydrogel: the non-dynamic hydrogel system (800. Mu.L) included 25mg/mL chitosan (500. Mu.L), 4mg/mL collagen (100. Mu.L), 5mg/mL genistein (100. Mu.L) and 6wt% beta-glycerophosphate (100. Mu.L). Wherein chitosan is dissolved in 0.1M acetic acid solution. Fully mixing and standing at 37 ℃ for 1h to form uniform chitosan/collagen non-dynamic hydrogel N-HP. The N-HPGO is obtained by adding 4 μg GDF-5 and 4 μg NMO during the preparation process.

Example 6 detection and application

The hydrogel state before and after the first cross-linking, scanning electron microscope SEM of the internal network of the dynamic hydrogel with/without ferrous ion treatment, drug release rate, biocompatibility test, important mechanical function, cell and living body treatment effect;

hydrogel state before and after the first cross-linking (fig. 3): under the action of the crosslinking agent, 1% sodium tetraborate, the uncrosslinked dynamic hydrogel rapidly crosslinked and produced a structural change.

SEM (fig. 4) of the internal network of dynamic hydrogels with/without ferrous ion treatment: scanning Electron Microscopy (SEM) was performed on a FEI Sirion 200 (operating at 30 kV) to observe the internal network of the dynamic hydrogels with/without ferrous ion treatment, as can be clearly seen under SEM, since the addition of GDF5 and Ori effectively improved the degree of cross-linking of the hydrogels by forming new dynamic bonds: compared with pure HP hydrogel, the pore size of HPGO is obviously reduced from 90.01 μm to 17.21 μm, and the porosity of HPGO is also reduced from 41.60% to 31.67%, which is reduced by 23.87%. In addition, the hydrogel after primary crosslinking is subjected to secondary crosslinking under the action of ferrous ions, the porosity of all dynamic hydrogels shows a decreasing trend, and compared with the original hydrogel sample, the pore size of HPGO with secondary crosslinking is reduced by 26.11%, and reaches 11.16 mu m. These changes in hydrogel porosity indicate that chelation with ferrous ions can enhance cross-linking between hydrogel precursors.

Binding behavior of hydrogels to ferrous iron was detected by raman spectroscopy and X-ray diffraction analysis (fig. 5): after treatment with ferrous ions, HPGO hydrogel has a Raman spectrum of 700-770cm ^-1 Characteristic peaks of Fe-O combination appear at the positions, and the success of iron coordination is directly indicated. To further verify the coordination between the dynamic hydrogel and ferrous ions, the ferrous ion treated hydrogels were measured by X-ray diffraction analysis and showed Fe at 32.9 ° and 51.6 °, respectively ²⁺ Is proved by the characteristic peak of the (2) that the dynamic hydrogel can bind Fe ²⁺ To reduce its excessive presence in the degenerative nucleus pulposus microenvironment.

Acidic controlled release of dynamic hydrogel-loaded drug (fig. 6): due to the inherent acid sensitivity of the schiff base bond between GDF5 and OHA, we incubated the dynamic hydrogel in biomimetic buffer at acidic pH and monitored the distribution of GDF 5. As shown in the figure, at each time point, the release amount of GDF5 in the buffer at pH5.5 was higher than that in the buffer at pH 7.4. At pH5.5, 65.88% of GDF5 was released in response to an acidic trigger within 48 hours, consistent with the pH responsiveness of the schiff base bond, and supporting our hypothesis that GDF5 could be released from the hydrogel matrix in an on-demand manner. The slow release case of Ori shows a similar trend as GDF 5. At 48 hours, the release of Ori in an acidic microenvironment at pH5.5 was 45.78%. This can be explained by the acid induced cleavage of schiff base bonds which can reduce the degree of cross-linking of the hydrogel matrix and promote diffusion of encapsulated Ori-containing nanovesicles. Furthermore, in HPGO with secondary cross-linking, the drug release functions were similar, notably that HPGO with secondary cross-linking released 58.64% of GDF in pH5.5 buffer responsively, 7.24% less than HPGO without secondary cross-linking, but without significant differences (p > 0.05). Similarly, ori release was reduced by 6.17% but there was no significant difference (p > 0.05). In combination with the previous porosity and pore size results, it is demonstrated that pH is a major factor affecting drug release properties of HPGO hydrogels compared to the secondary cross-linking induction effect.

48-hour biocompatibility test of dynamic hydrogels (fig. 7):

cell viability test: 10% by volume CCK-8 solution was added to the medium, and after three hours incubation, the absorbance of CCK-8 in the supernatant was measured at 450nm using a microplate reader (BioTek, USA) to assess the activity of NPC; the left figure is a schematic representation of co-culture of hydrogel and nucleus pulposus cells, the hydrogel being immersed in a culture medium together with the cells through a scaffold. The right panel shows the results of the cell activity assay for co-culture, with no significant difference in NPC activity for dynamic hydrogel co-culture compared to the control group without hydrogel co-culture (p > 0.05).

Mechanical characteristics of dynamic hydrogels (fig. 8):

the measurement mode is as follows: measurement of compression Properties: each hydrogel sample was processed into a cylindrical shape with a diameter of 4mm and a height of 4mm, and immersed in distilled water or a ferrous ion solution, respectively, for 2 hours. The hydrogel was compressed to a height of 30% by a 30N sensor at a rate of 0.05mm/s using a small multi-scale in situ mechanical testing system IBTC-300. The compression modulus is calculated from the following equation:

wherein epsilon and sigma represent strain and stress, respectively. L, lo, P, A represent the deformed length of the hydrogel sample, the original pitch length of the hydrogel sample, the compressive load, and the initial cross-sectional area of the hydrogel sample, respectively.

The results illustrate: maintaining elasticity and compression resistance are one of the most important physiological functions of the intervertebral disc, and the compression resistance of the hydrogel often determines the treatment effect of the hydrogel on the intervertebral disc, so that the compression performance is the most important mechanical characteristic of the hydrogel, and is often quantitatively evaluated through compression modulus. Because the dynamic hydrogel is rich in dynamic bonds (acylhydrazone bonds, imine bonds, hydrogen bonds and the like), the dynamic bonds of the contact surface can be crosslinked again after two broken ends are contacted so as to achieve self-healing.

The upper diagram is a schematic diagram, the left is a schematic diagram of a measurement mode, the middle is the crosslinking effect of two dynamic hydrogels, the contact surface is obviously crosslinked, and the right is a schematic diagram thereof

The lower graph shows the measurement results, the left graph shows the primary crosslinking and the reaction between Fe ²⁺ As a result of the compression performance after the ion-induced secondary crosslinking, the dynamic hydrogel structure is further crosslinked after the secondary crosslinking, so that the compression performance is better, and compared with the primary crosslinking, the compression modulus of HP/HPG/HPO/HPGO is respectively improved by 34.6%/92.9%/172.4%/125.7%; compared with HP/HPG/HPO, HPGO has the advantages that more dynamic bonds are formed due to the carrying of more drugs, so that the compression performance is enhanced, the compression modulus of HPGO after primary and secondary crosslinking is respectively 9.17kPa and 20.73kPa, and 58.9% and 177.68% are respectively increased compared with HP; since the dynamic bond hydrogels self-heal, the compression modulus of the self-healing HP/HPG/HPO/HPGO after primary and secondary crosslinking was reduced by 39.7%/52.6%/11.1%/21.2% and 15.4%/51.3%/39.1%/57.8%, respectively. The right graph shows the compression performance results of the basic hydrogel HP, the 2nd-HP and the non-dynamic hydrogel N-HP after secondary crosslinking and the non-dynamic hydrogel N-HPGO loaded with drugs, and the compression performance after damage is drastically reduced by about 79.6%/77.1% because the N-HP/N-HPGO cannot form new dynamic bonds.

Cell viability assay after 48 hours of co-culture of nucleus pulposus cells with hydrogel under inflammation induction (fig. 9):

the left graph shows that the four hydrogels and the nucleus pulposus cells contained in the invention are co-cultured, and the three hydrogels HPG/HPO/HPGO loaded with medicines have remarkable treatment effect under the condition of inflammation induced by TBHP, and the treatment effect of the two medicines is best, and the cell activity is improved to 94.7 percent (p < 0.05) compared with 52.4 percent of that of a control group. The right graph shows that the basic hydrogel HP and the hydrogel HPGO with the best treatment effect and the non-dynamic hydrogel N-HP of the invention simply take the medicine GO (100 ng/ml), the non-dynamic hydrogel carries the medicine N-HPGO and the nucleus pulposus cells to co-culture, and the same, the pure medicine and the hydrogel carried with the medicine have obvious treatment effects, and the sustained release effect of the HPGO can be seen that the treatment effect is superior to that of the pure medicine and the non-dynamic hydrogel N-HPGO, and the cell activity is increased by 15.8 percent and 19.2 percent (p < 0.05) compared with that of the N-HP/N-HPGO.

Therapeutic effects of different hydrogels after rat tail disc puncture (fig. 10):

the upper graph shows the therapeutic effect of four hydrogels contained in the present invention in a rat tail intervertebral disc puncture model

And (3) a molding process: rats at week 8 were anesthetized with 21G needles penetrating the Co7/8 and Co8/9 discs at the proximal end of the tail, rotated 360 degrees and kept inserted for 15 seconds, three days post-operatively each experimental group was injected with a different 10 μl hydrogel treatment, and discs were harvested at week 6 post-operation and cut into slices.

Description of results: the base hydrogel HP can obviously reduce the degeneration degree of the nucleus pulposus due to the excellent mechanical supporting effect of the dynamic hydrogel, and the HPG/HPO/HPGO can slowly release the medicine on the basis, so that the dynamic hydrogel has different degrees of treatment effects on the nucleus pulposus tissue, wherein the HPGO has the most obvious treatment effect. Compared with the operation control group, the HPGO group nucleus pulposus tissue HE staining can obviously observe the cell staining and the maintenance of normal nucleus pulposus morphology, which suggests that the HPGO treatment improves the reduction of nucleus pulposus cell quantity caused by operation, while in SO/FG staining, the red staining of the HPGO group nucleus pulposus area is obviously better, which indicates that more extracellular matrix exists in the HPGO group nucleus pulposus tissue, and meanwhile, the green staining is less, which indicates that the HPGO group nucleus pulposus tissue is fibrosed or mineralized less. HPGO treatment was suggested to restore the function of nucleus pulposus cells.

The following graph shows the therapeutic effects of the basic hydrogel HP, the hydrogel HPGO with the best therapeutic effect and the non-dynamic hydrogel N-HP, the pure administration GO and the non-dynamic hydrogel carried drug N-HPGO in a rat model: because of the mechanical defect of N-HP, the treatment effect in the rat puncture model is not obvious, and the defects are also generated in the N-HP/N-HPGO, specifically, the N-HP/N-HPGO group is obviously inferior to the HPGO group in staining by HE, and the nucleus pulposus morphology is changed to a certain extent; in the SO/FG staining, the N-HP/N-HPGO also has the defects of poor red staining and more green staining, which suggests that the nuclear tissue cannot be recovered well, and part of the nuclear tissue is replaced by fibrous tissue or mineralized tissue. In conclusion, animal experiment results prove that the hydrogel has excellent treatment effect in intervertebral disc treatment.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims (10)

1. An environmentally-friendly dynamic hydrogel, wherein the hydrogel comprises OHA, PVA, and sodium tetraborate; NMO or GDF-5.

2. The environmentally-adapted dynamic hydrogel according to claim 1, wherein the NMO is prepared by the method comprising: extracting nucleus pulposus cell membrane (nano vesicle NM) by repeated extrusion by adopting a liposome extrusion method, mixing the extracted NM with Ori in a volume ratio of 2:1 in a liposome extruder, and forming NMO by repeated extrusion.

3. The environmentally-friendly dynamic hydrogel according to claim 1, wherein the Oxidized Hyaluronic Acid (OHA) is prepared by the following method: 10mg of Hyaluronic Acid (HA) was dissolved in 100mL of distilled water, followed by the addition of 20mM sodium periodate; the mixture was stirred for 24 hours in the dark; thereafter, the mixture was dialyzed in the dark (dialysis bag MW 3500 Da) for 3 days and dried under vacuum to obtain aldehyde group-containing Oxidized Hyaluronic Acid (OHA) for the subsequent experiments.

4. The environmentally-adapted dynamic hydrogel according to claim 1, wherein the OHA concentration is 5-15mg/mL, the PVA concentration is 5-15% w/v, and the sodium tetraborate concentration is 0.5-1.5% w/v.

5. The environmentally-adapted dynamic hydrogel according to claim 1, wherein the OHA concentration is 10mg/mL, the PVA concentration is 10% w/v, and the sodium tetraborate concentration is 1% w/v.

6. The preparation method of the environment-adaptive dynamic hydrogel is characterized by comprising the following steps of: (1) Preparation and detection of Ori-loaded Nanovesicles (NMO); (2) Preparing and detecting hydrogel precursors, namely Oxidized Hyaluronic Acid (OHA); (3) preparation and detection of dynamic hydrogel.

7. The method for preparing an environmentally-friendly dynamic hydrogel according to claim 6, wherein the preparing of NMO comprises: extracting nucleus pulposus cell membrane (nano vesicle NM) by repeated extrusion by adopting a liposome extrusion method, mixing the extracted NM with Ori in a volume ratio of 2:1 in a liposome extruder, and forming NMO by repeated extrusion.

8. The method of preparing an environmentally-friendly dynamic hydrogel according to claim 6, wherein the preparation of Oxidized Hyaluronic Acid (OHA) comprises: 10mg of Hyaluronic Acid (HA) was dissolved in 100mL of distilled water, followed by the addition of 20mM sodium periodate; the mixture was stirred for 24 hours in the dark; thereafter, the mixture was dialyzed in the dark (dialysis bag MW 3500 Da) for 3 days and dried under vacuum to obtain aldehyde group-containing Oxidized Hyaluronic Acid (OHA) for the subsequent experiments.

9. The method for preparing an environmentally-friendly dynamic hydrogel according to claim 6, wherein the preparation of the dynamic hydrogel comprises: 10mg of OHA was dissolved in 100mL of PBS (pH=7.4) to obtain 10mg/mL of OHA solution; meanwhile, 1g of polyvinyl alcohol (PVA) was dissolved in 10mL of PBS (ph=7.4) at 90 ℃ to obtain a 10% PVA solution; mixing 300. Mu.L 10% PVA solution and 700. Mu.L OHA solution (10 mg/mL) at room temperature; the mixture was then added to 1% sodium tetraborate to immediately initiate the formation of a composite hydrogel, designated HP, at room temperature; similarly, GDF5, NMO and GDF5+NMO were added to a mixture of 300. Mu.L 10% PVA solution and 700. Mu.L OHA solution (10 mg/mL) to form HPG, HPO and HPGO hydrogels, respectively, after the introduction of 1% sodium tetraborate.

10. Use of an environmentally-adapted dynamic hydrogel according to any one of claims 1 to 5 or a method of preparing an environmentally-adapted dynamic hydrogel according to any one of claims 6 to 9, for a) preparing a reagent or kit for use in bioengineering; or b) preparing a reagent or a kit for repairing or reconstructing the tissue of the intervertebral disc; or c) preparing a medicament for treating intervertebral discs; or d) a method of restoring the function of a nucleus pulposus cell, optionally for non-diagnostic or non-therapeutic purposes.