Use of a viscoelastic biomaterial

Abstract

The application discloses application of a viscoelastic biomaterial in the technical field of biological medicine, and aims to solve the problem of the influence mechanism of viscoelasticity on the metabolic state and pathway of nucleus pulposus cells in the prior art. The application comprises application of a viscoelastic double-network hydrogel of the viscoelastic biomaterial as a drug for promoting tissue repair and application of the viscoelastic biomaterial as a therapeutic drug for intervertebral disc degeneration; the application is suitable for development of a tissue repair drug, and a tissue repair drug is prepared from the viscoelastic biomaterial, so that a new route is provided for development of a tissue repair preparation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and to the application of a viscoelastic biomaterial, particularly to the application of a viscoelastic dual-network hydrogel in the preparation of drugs that promote tissue repair and drugs that treat intervertebral disc degeneration. Background Technology

[0002] The regulatory role of external mechanical cues in cell fate is increasingly recognized. Cells regulate their biochemical functions by sensing mechanical stimuli from the matrix, thereby influencing key biological processes such as cell growth, proliferation, migration, and differentiation. The extracellular matrix (ECM) is a complex, dynamic, and cross-linked network structure containing tethered biomolecules. It forms the basis of the morphology and function of tissues and organs, providing crucial physical support and biomechanical signals necessary for tissue development. Therefore, manipulating cell-ECM interactions by regulating the physical properties of the ECM has become a potential strategy for intervening in the occurrence and development of diseases. Viscoelasticity is a common mechanical characteristic of living tissues and the ECM. Unlike the matrix elastic modulus, viscoelasticity reflects the combined characteristics of tissue binding elasticity and viscous behavior. Purely elastic materials can completely store, convert, and release input energy, following Hooke's law, with stress and strain occurring simultaneously without any energy loss; this can be characterized by the storage modulus. In contrast, purely viscous materials completely dissipate and lose input energy through internal friction, following Newton's laws of fluid dynamics; this can be characterized by the loss modulus. Most biological tissues possess viscoelastic properties that fall between pure elasticity and pure viscosity, enabling them to adapt to their complex biological functions. However, previous biomaterial designs have often neglected to consider the viscoelasticity of biological scaffolds.

[0003] Intervertebral disc degeneration (IVDD) is the main pathological basis of low back pain, characterized by the progressive structural and functional failure of nucleus pulposus cells (NPCs). As a shock-absorbing structure connecting adjacent vertebrae, the nucleus pulposus, under physiological conditions, contains abundant proteoglycans, maintaining its highly hydrated and viscoelastic mechanical characteristics. It can undergo rapid deformation and stress relaxation under repeated shear stress, maintaining spinal biomechanical balance through energy storage and pressure load redistribution. In the process of IVDD, the aging nucleus pulposus tissue exhibits downregulation of proteoglycan secretion, collagen structure imbalance, and a tendency towards fibrosis. Viscoelasticity may be lost, weakening its shock-absorbing capacity against axial pressure. This deterioration of the biomechanical microenvironment not only weakens the biomechanical function of the intervertebral disc but also profoundly affects the cell survival, phenotype, and metabolic activity of nucleus pulposus cells through mechanotransduction pathways, exacerbating the disease. However, current biomaterial therapy strategies for IVDD primarily focus on modulating the intervertebral disc microenvironment, neglecting the crucial guiding role of ECM viscoelasticity in its development and disease progression. Therefore, research into the role and mechanism of tissue viscoelasticity imbalance in the intervertebral disc degeneration process is significant and can guide the design of next-generation biomaterials.

[0004] Over the past decade, the ability of metabolism to guide cell fate has received considerable attention. Cellular metabolism not only contributes to energy production but also plays a crucial role in cell proliferation and lineage determination. Nucleus pulposus cells, as the core executors maintaining ECM homeostasis, influence the balance between the synthesis and catabolism of biomolecules such as type II collagen and proteoglycans through their energy metabolism. The ECM is both a product of nucleus pulposus cell metabolism and can provide multidimensional cues to nucleus pulposus cells by constructing the microenvironment, enabling precise regulation of cellular metabolism. After sensing the physical cues provided by the ECM, cells trigger the transduction of biochemical signals through mechanotransduction, thereby achieving metabolic regulation at the molecular signaling level. ECM stiffness maintains and regulates nucleus pulposus cell glucose metabolism and ATP production by modulating the activity and cellular localization of myocardin-associated transcription factor-A (MRTF-A), while the 3D topology of the ECM guides the state of glycolysis and oxidative phosphorylation in nucleus pulposus cells by regulating mitochondrial dynamics. Therefore, the extracellular matrix (ECM) plays a central role in cellular bioenergetics through physically driven mechano-biochemical signal transduction, and is an important mechanism for maintaining the metabolic function of nucleus pulposus cells and intervertebral disc homeostasis. Viscoelasticity, a property universally possessed by living cells and tissues, is a core physical property of the extracellular matrix. It works synergistically with biochemical signals to form a "mechanical-biochemical" coupled signal network that guides cell fate.

[0005] Viscoelasticity is a common mechanical characteristic of living tissues and ECMs. However, current technologies lack research on the mechanical transduction mechanism of tissue viscoelasticity in regulating the metabolic state of NPCs. When designing tissue repair drugs and biological scaffolds, the core guiding role of ECM viscoelasticity in their occurrence, development and disease outcome has been neglected. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a viscoelastic dual-network hydrogel, revealing the mechanotransduction mechanism by which tissue viscoelasticity regulates cellular metabolic state, and providing a new direction for the development of tissue repair drugs.

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0008] In a first aspect, the present invention provides the application of a viscoelastic dual-network hydrogel as a drug for promoting tissue repair.

[0009] This invention constructs three types of cell-carrying hydrogel microspheres with differential viscoelasticity and injects them in situ into the rat caudal intervertebral disc. The results demonstrate that the rapidly relaxing matrix hydrogel microspheres significantly increase the ECM content in the nucleus pulposus tissue, effectively restoring disc height and MRI signal, suggesting promising applications in tissue regeneration. In summary, the data from this invention indicate that matrix viscoelasticity, as one of the most important physical properties of the ECM, is a core driving force for achieving the coupling of micromechanical regulation of cell metabolism and physiological function.

[0010] Optionally, the tissue is nucleus pulposus tissue.

[0011] Furthermore, the viscoelastic dual-network hydrogel promotes tissue repair by enhancing cellular glycolysis through a viscoelastic matrix.

[0012] Furthermore, the viscoelastic dual-network hydrogel promotes tissue repair by increasing the extracellular matrix content in the tissue through a viscoelastic matrix.

[0013] Optionally, the viscoelastic dual-network hydrogel is a porous hydrogel with a rapid stress relaxation matrix.

[0014] Furthermore, the preparation of the viscoelastic dual-network hydrogel includes the following steps:

[0015] Sodium periodate was added to a hyaluronic acid solution and stirred under dark conditions to modify it. The modified solution was then post-treated to obtain aldehyde-modified hyaluronic acid.

[0016] Aldehyaluronic acid was dissolved, methacrylate was added to the aldehyde-modified hyaluronic acid solution, the pH of the solution was adjusted to 8-8.5, the pH-adjusted solution was placed in an ice bath to carry out the substitution reaction, and the solution after the substitution reaction was post-treated to obtain aldehyde- and methacryloyl-modified hyaluronic acid.

[0017] Aldehyde and methacryloyl-modified hyaluronic acid were dissolved in a photoinitiator solution, and bovine collagen powder was added to the dissolved solution to obtain a hydrogel precursor solution.

[0018] Bulk viscoelastic dual-network hydrogels or viscoelastic dual-network hydrogel microspheres were prepared using hydrogel precursor solutions.

[0019] Furthermore, the steps for preparing bulk viscoelastic dual-network hydrogels from hydrogel precursor solutions include: placing the hydrogel precursor solution under 365 nm ultraviolet light irradiation to obtain bulk viscoelastic dual-network hydrogels.

[0020] Furthermore, the steps for preparing viscoelastic dual-network hydrogel microspheres from the hydrogel precursor solution include: preparing microspheres from the hydrogel precursor solution using a water-in-oil microfluidic method, and post-processing the microspheres to obtain viscoelastic dual-network hydrogel microspheres.

[0021] Optionally, the mass ratio of hyaluronic acid to sodium periodate is 20:11.

[0022] Optionally, the mass-to-volume ratio of aldehyde-modified hyaluronic acid to methacrylate is 1:1.

[0023] Furthermore, the mass ratio of the photoinitiator to the aldehyde and methacryloyl-modified hyaluronic acid is 1:8.

[0024] Optionally, the post-treatment of the modified solution includes the following steps:

[0025] Ethylene glycol was added to the modified solution for 1 hour to deactivate the product.

[0026] The modified product was packaged into a dialysis bag and immersed in ultrapure water for dialysis for 3 days, with the ultrapure water being changed daily.

[0027] The modified product after dialysis was freeze-dried to obtain aldehyde-modified hyaluronic acid.

[0028] Optionally, the post-treatment of the substitution reaction product includes the following steps:

[0029] The substitution reaction product was encapsulated in a dialysis bag and then immersed in ultrapure water for dialysis for 3 days, with the ultrapure water being changed daily.

[0030] The dialysis product of the substitution reaction was freeze-dried to obtain hyaluronic acid with dual modification of aldehyde and methacryloyl groups.

[0031] Optionally, the post-processing of the microspheres includes the following steps:

[0032] Microspheres were irradiated with ultraviolet light.

[0033] The cross-linked hydrogel microspheres irradiated with ultraviolet light were washed three times with 75% ethanol and five times with PBS. The PBS was replaced every three hours to obtain the purified microspheres.

[0034] The purified microspheres were frozen overnight at -80°C, and then freeze-dried to obtain viscoelastic double-network hydrogel microspheres.

[0035] Secondly, the present invention provides the application of a viscoelastic dual-network hydrogel as a therapeutic agent for intervertebral disc degeneration.

[0036] Beneficial effects

[0037] This invention prepares a cell-loaded viscoelastic dual-network hydrogel and injects it into the intervertebral disc in situ, implanting the mechanical transduction effect of the highly viscoelastic matrix into the tissue. It verifies that the rapidly stress-relaxed matrix also has multiple effects in vivo, such as promoting cell proliferation, inhibiting cell apoptosis, improving inflammatory response and extracellular matrix accumulation, demonstrating that viscoelastic biomaterials have the application prospect as tissue repair drugs and treatment drugs for intervertebral disc degeneration. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 These are the test results of hyaluronic acid modification in Example 1 of the present invention and the characterization results of hydrogel in Example 3 of the present invention, wherein... Figure 1 Image A represents the 1H NMR spectrum analysis result of the modified hyaluronic acid. Figure 1 Image B represents the Fourier transform infrared spectroscopy analysis results of modified hyaluronic acid. Figure 1 C represents the microstructure observation (scale bars, 200 μm) of three hydrogels with increasing dynamic bond content under AFM (atomic force microscopy). Figure 1 D represents the SEM microstructure observation results (scale bars, 200 μm and 50 μm) of three hydrogels with increasing dynamic bond content;

[0040] Figure 2 The figures shown are of the porosity analysis, rheological properties testing, and compression deformation testing results of the hydrogel in Example 3 of this invention. Figure 2 Figure A shows the porosity analysis results for the three types of hydrogels. Figure 2 Rheological analysis of three hydrogels with increasing dynamic bond content is shown in section B. Figure 2 In the middle, C represents the stress-relaxation behavior of three hydrogels with increasing dynamic bond content. Figure 2 D represents the τ values ​​of three hydrogels with increasing dynamic bond content. 1 / 2 Time quantification, Figure 2 E represents the macroscopic compression curves of three hydrogels with increasing dynamic bond content. Figure 2 F represents the statistical analysis result of Young's modulus obtained from the compression curve;

[0041] Figure 3 This is a graph showing the cell compatibility test results of the differentially viscoelastic hydrogel of Example 4 of the present invention, wherein... Figure 3 Image A shows images of nucleus pulposus cells in a co-culture system observed under an optical microscope at 2 hours, 8 hours, and 24 hours (Scale bar, 100 μm). Figure 3 Image B shows the cytoskeleton staining image (Scale bar, 50 μm) 3 days after co-culture. Figure 3 In the middle (C), the roundness of nucleus pulposus cells was quantitatively analyzed based on cytoskeleton staining in a co-culture system. Figure 3 Image D shows live / dead cell staining and EdU cell proliferation assay images after 3 days of co-culture (Scale bars, 400 μm and 100 μm). Figure 3 E represents the quantitative analysis of EdU cell proliferation assay. Figure 3 The data in section F are Ki-67 cells after 3 days of co-culture analysis by flow cytometry. Figure 3 G represents a quantitative analysis of the Ki-67 positivity rate;

[0042] Figure 4 This is a diagram showing the tissue compatibility test results of the differentially viscoelastic hydrogel of Example 5 of the present invention, wherein... Figure 4 Image A shows the H&E images (scale bars, 2 mm and 200 μm) of the implanted hydrogel in rats 7 and 14 days after subcutaneous implantation. Figure 4 Image B shows H&E staining images (Scale bar, 200 μm) of the heart, liver, spleen, lungs, and kidneys of rats in each group after subcutaneous implantation.

[0043] Figure 5 This is a graph showing the metabolomics and transcriptomics analysis results of Example 6 of the present invention, wherein... Figure 5 China A Figure 5 China B and Figure 5 The results from F show the differences in cell metabolites in the matrix at different stress relaxation rates; Figure 5 Analysis of the content of C in the middle is fructose-6-phosphate. Figure 5 D represents the content analysis of sodium lactate. Figure 5 E represents the content analysis of pyruvate;

[0044] Figure 6 This is a graph showing the Seahorse metabolic analysis results of Example 6 of the present invention, wherein... Figure 6 Figure A shows the extracellular acidification rate curves of the hippocampus in the three groups. Figure 6 B represents the quantitative analysis results of the basal glycolysis level in the three groups. Figure 6 C represents the quantitative analysis result of glycolytic capacity. Figure 6 The results of quantitative analysis of glycolysis reserves are shown in D.

[0045] Figure 7 This is a graph showing the detection results of oxidative stress levels in nucleus pulposus cells in Example 6 of the present invention, wherein... Figure 7 Image A shows JC-1 stained images (scale bar, 50 μm) of mitochondrial membrane potential in nucleus pulposus cells co-cultured with hydrogels containing three differentially viscoelastic matrices: SLOW, MEDIUM, and FAST. Figure 7 B represents the quantitative analysis results of JC-1 staining. Figure 7 C represents the mitochondrial morphology observation results of the differential viscoelastic matrix hydrogel group. Figure 7 D represents the DCFH-DA analysis results of nucleus pulposus cells after co-culturing with hydrogels containing differentially viscoelastic matrices. Figure 7 E in the middle represents the results of MitoSOX flow cytometry analysis;

[0046] Figure 8 This is a graph showing the detection results of apoptosis levels in Example 6 of the present invention, wherein... Figure 8 In Figure A, Annexin V-FITC / PI was used to detect the apoptosis level in nucleus pulposus cells. Figure 8 Image B shows the experimental results of hydrogel TUNEL staining with three different stress relaxation rates. Figure 8 C represents the quantitative analysis results of TUNEL staining;

[0047] Figure 9 These are characterization and analysis results of the three types of hydrogel microspheres in Example 8 of the present invention, wherein... Figure 9 Image A shows the microstructure results of FMS, SMS, and MMS observed under SEM. Figure 9 Image B shows the microstructure and immunofluorescence staining results of three hydrogel microspheres with different viscoelasticities observed under SEM. Figure 9 Image C shows the immunofluorescence images of type II collagen and proteoglycan in three types of cell-carrying hydrogel microspheres. Figure 9 D in the image represents the quantitative analysis results of the immunofluorescence image;

[0048] Figure 10 This is an image of the small animal imaging result of Embodiment 9 of the present invention, wherein, Figure 10 Image A shows small animal imaging of three types of cell-carrying hydrogel microspheres in the rat tailbone environment. Figure 10 Quantitative analysis of fluorescence intensity in small animal imaging images at 0, 3, 5, 7, and 14 days (B) was performed.

[0049] Figure 11 This is an image showing the imaging evaluation results of the animal specimens in Example 9 of the present invention, wherein... Figure 11 Image A shows X-ray and T2-weighted MRI images of the rat caudal vertebrae within each group at weeks 4 and 8 after the animal model was established. Figure 11 In section B, a quantitative analysis of the changes in intervertebral disc height index (DHI%) was performed on the five groups at 4 and 8 weeks. Figure 11 C represents the quantitative analysis of MRI grading at 4 weeks based on T2-weighted MRI images. Figure 11 The middle D is a quantitative analysis of MRI grading at 8 weeks using T2-weighted MRI images;

[0050] Figure 12 This is a histological evaluation result diagram of the animal specimen of Example 9 of the present invention, wherein, Figure 12 Image A shows tissue H&E images and red-to-green images for each group at 4 and 8 weeks. Figure 12 Figure B represents the histological grading and quantitative analysis performed at 4 weeks and 8 weeks after 4 punctures, based on tissue sections. Detailed Implementation

[0051] The above content is further illustrated below with specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments. All technologies implemented based on the above content of this invention fall within the scope of this invention.

[0052] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.

[0053] The statistical analysis data in the following examples are presented in the form of mean ± standard deviation. Statistical analysis was performed using one-way ANOVA or Tukey's test, and statistical significance was set as P < 0.05.

[0054] Table 1: Experimental materials.

[0055]

[0056] Example 1

[0057] This embodiment provides a method for preparing modified hyaluronic acid. The synthesis and characterization of the double-modified hyaluronic acid are shown in the figure below. Figure 1 As shown, it includes the following steps:

[0058] Aldehyde acid (HA) is modified by aldehyde grouping:

[0059] 1 g of solid HA was dissolved in 100 ml of ultrapure water and stirred overnight to ensure complete dissolution. 5 ml of 0.5 mol / L sodium periodate was added to the HA solution, and the mixture was stirred at 250 rpm in the dark for 2 hours at room temperature. Then, 1 ml of ethylene glycol was added to inactivate unreacted periodate for 1 hour, yielding the product. The product was encapsulated in a dialysis bag and immersed in ultrapure water for dialysis for 3 days, with the ultrapure water being changed daily. The product in the dialysis bag was then freeze-dried to obtain aldehyde-modified hyaluronic acid (AHA).

[0060] HA was modified by methacrylate esterification:

[0061] 1 g of HA was dissolved in 100 ml of ultrapure water, stirring constantly until completely dissolved. Then, 1 ml of methacrylate was added to the HA solution to adjust the pH to 8.5. The solution was then placed on ice for the substitution reaction, maintaining an ice bath for 12 hours. After the reaction was complete, the product was obtained. The product was encapsulated in a dialysis bag and immersed in ultrapure water for dialysis for 3 days, with the external ultrapure water changed daily. The product in the dialysis bag was freeze-dried to obtain methacrylamide hyaluronic acid (HAMA).

[0062] Methacrylate modification of AHA:

[0063] 1g of AHA was dissolved in 100ml of ultrapure water, stirring constantly until completely dissolved. Then, 1ml of methacrylate was added to the AHA solution to adjust the pH to 8.5. The solution was then placed on ice for the substitution reaction, maintaining an ice bath for 12 hours. After the reaction was complete, the product was obtained. The product was encapsulated in a dialysis bag and immersed in ultrapure water for dialysis for 3 days, with the external ultrapure water being changed daily. The product in the dialysis bag was freeze-dried to obtain hyaluronic acid (AHAMA) dual-modified with aldehyde and methacryloyl groups.

[0064] Detection of modified hyaluronic acid:

[0065] HA, AHA, HAMA, and AHAMA were dissolved using deuterated heavy water (D2O), respectively. 1¹H NMR spectra of the four substances were measured and their composition analyzed. Subsequently, Fourier transform infrared spectroscopy was used to verify the success of the modification. The detection results for hyaluronic acid modification are shown below. Figure 1 , Figure 1 Image A represents the 1H NMR spectrum analysis result of the modified hyaluronic acid. Figure 1 Figure B shows the Fourier transform infrared (FTIR) analysis results of the modified hyaluronic acid. The results indicate that significant nascent doublets were observed at 5.6 ppm and 6.0 ppm in the AHAMA and HAMA groups, corresponding to the C=C double bond in methacrylate, confirming that the hyaluronic acid underwent methacrylation. Furthermore, an additional nascent singlet was observed at 4.9 ppm in both the AHA and AHAMA groups. This can be interpreted as a nascent peak formed by the aldehyde group and the adjacent hemiacetal proton, confirming the successful introduction of the aldehyde group in the AHA and AHAMA groups. The C=O stretching vibration peak at 1710 cm⁻¹ in the FTIR spectra of AHAMA and HAMA further confirms the successful methacrylation.

[0066] Example 2

[0067] This embodiment provides a method for preparing a viscoelastic hydrogel, including the following steps:

[0068] First, lithium phenyl-2,4,6-trimethylbenzoyl phosphate was dissolved in 10 ml of PBS at a concentration of 0.25% (w / v). Then, 200 mg of AHAMA was weighed and added to 10 ml of PBS until completely dissolved. Next, bovine collagen powder was added to the solution to achieve a final concentration of 3%. After the bovine collagen powder was completely dissolved, a hydrogel precursor solution was obtained. This hydrogel precursor solution was added to a gel mold, and the gel was then subjected to 365 nm ultraviolet light (10 mW / cm²). 2 After curing for 5 minutes, a block hydrogel is formed, resulting in 2% AHAMA hydrogel, or simply 2% AHAMA / COL.

[0069] Comparative Example 1

[0070] This comparative example provides a method for preparing a viscoelastic hydrogel. Under the same conditions as in Example 2, 200 mg AHAMA was replaced with 200 mg HAMA to obtain a 2% HAMA hydrogel, which is abbreviated as 2% HAMA / COL.

[0071] Comparative Example 2

[0072] This comparative example provides a method for preparing a viscoelastic hydrogel. Under the same conditions as in Example 2, 200 mg AHAMA is replaced with 100 mg HAMA + 100 mg AHAMA to obtain a 1% HAMA / 1% AHAMA hydrogel, abbreviated as 1% HAMA / 1% AHAMAA / COL.

[0073] Example 3

[0074] This embodiment provides a characterization and detection method for three hydrogels from Example 2, Comparative Example 1, and Comparative Example 2, including the following:

[0075] The surface morphology and surface roughness of three groups of hydrogels were measured using atomic force microscopy in Tapping mode.

[0076] Three types of hydrogels were fixed onto conductive adhesives and sputtered with gold using an ion sputtering apparatus. The samples were then observed using a Hitachi S-4800 scanning electron microscope (SEM). The accelerating voltage was adjusted to 10 kV. During SEM observation, the porosity of the three groups of hydrogels was measured based on the obtained SEM microscopic images.

[0077] The results of the characterization and testing of the hydrogel are shown in Figure 1 , Figure 1 C represents the microstructure observation (scale bars, 200 μm) of three hydrogels with increasing dynamic bond content under AFM (atomic force microscopy). The surface roughness Ra values ​​of the three hydrogels are 8.47, 7.31 and 7.62, respectively, with no significant difference. Figure 1 Figure D shows the SEM microstructure observations (scale bars, 200 μm and 50 μm) of three hydrogels with increasing dynamic bond content. The porosity analysis results of the three hydrogels are as follows: Figure 2 As shown in Figure A, the results indicate that there is no significant difference in pore size among the three types of gels.

[0078] To test the rheological properties of the hydrogels, a rotational rheometer was used to measure the rheological properties of the three hydrogels. Three cylindrical block hydrogels, prepared in a mold, were transferred to the middle of an 8 mm parallel plate with appropriate gaps. The angular momentum during strain scanning was set to 10 rad / s, and the levels of G′ and G″ of the three gels were measured at different time points. To verify the stress relaxation behavior of the three hydrogels, stress-relaxation tests were performed using a rotational rheometer. An initial deformation of 5% was set for the hydrogel block, and the measurement time lasted for 1000 s. After obtaining the stress-relaxation curves, the half-stress-relaxation time (τ) was calculated. 1 / 2 time).

[0079] For macroscopic compression deformation experiments of hydrogels, a general-purpose mechanical testing machine was used. The hydrogels were prepared in cylindrical or dumbbell-shaped containers, pretreated in PBS for 12 hours, and then subjected to compression tests. The compressive modulus of the hydrogel was obtained by calculating the slope of the stress-strain curve passing through the origin.

[0080] The results of hydrogel rheological property testing and compression deformation testing are shown in Figure 2 , Figure 2 Rheological analysis of three hydrogels with increasing dynamic bond content is shown in section B. Figure 2 In the middle, C represents the stress-relaxation behavior of three hydrogels with increasing dynamic bond content. Figure 2 D represents the τ values ​​of three hydrogels with increasing dynamic bond content. 1 / 2 Quantitative analysis over time showed that the stress relaxation rate of 2% AHAMA / COL was significantly shortened, and rapid relaxation performance was obtained through the introduction of dynamic chemical bonds. Figure 2 E represents the macroscopic compression curves of three hydrogels with increasing dynamic bond content. Statistical analysis of the Young's modulus obtained from the compression curves is as follows: Figure 2 As shown in Figure F, the three exhibit similar Young's moduli in macroscopic compression experiments.

[0081] Example 4

[0082] This embodiment provides a cell compatibility test for three hydrogels with different viscoelasticities, namely Example 2, Comparative Example 1, and Comparative Example 2, including the following:

[0083] The differential viscoelastic hydrogels described correspond to the 2% AHAMA / COL of Example 2, which has more dynamic bonds and is a hydrogel for rapid stress relaxation matrix, labeled as FAST in the figure; the 1% HAMA / 1% AHAMAA / COL of Comparative Example 2, which has moderate dynamic bonds and is a hydrogel for medium-speed stress relaxation matrix, labeled as MEIDUM in the figure; and the 2% HAMA / COL of Comparative Example 1, which has fewer dynamic bonds and is a hydrogel for slow stress relaxation matrix, labeled as SLOW in the figure.

[0084] After the preparation of the three hydrogel precursors in Example 2, Comparative Example 1, and Comparative Example 2, the hydrogel precursor solutions were aspirated using a sterile syringe and filtered through a 220 nm filter to remove bacteria. Cell crawling sheets were then placed at the bottom of a 24-well plate, and 200 μl of the filtered hydrogel precursor solution was added to each well. The precursor was then immediately cross-linked using a UV lamp. After washing three times with PBS, cell suspensions were inoculated, and the cells were allowed to adhere for 24 hours to establish a co-culture system of nucleus pulposus cells and differentially viscoelastic hydrogels. The cells were observed directly under an optical microscope at 2, 8, and 24 hours post-inoculation. During inoculation, it was difficult to ensure that the size and shape of the hydrogel, cured by UV irradiation, matched the wells in the 24-well plate. Therefore, placing the hydrogel precursor into the wells first and then curing it with UV irradiation resulted in a better fit and facilitated the operation.

[0085] The results of the hydrogel's cell compatibility test are as follows: Figure 3 As shown. Images of nucleus pulposus cells in the co-culture system observed under a light microscope at 2 hours, 8 hours, and 24 hours (Scale bar, 100 μm). Figure 3 As shown in Figure A, the results indicate good biocompatibility among the three types of hydrogels. In the FAST gel-based group, the number of spindle-shaped nucleus pulposus cells significantly increased. 72 hours after inoculation, the culture medium from the three different hydrogel co-culture systems was aspirated, washed three times with PBS, and then 4% paraformaldehyde was added. The cells were fixed at room temperature for 30 minutes. After drilling with a Triton X-100, the cells were blocked with 5% bovine serum albumin solution and incubated overnight at 4°C. After washing with PBS, phalloidin and DAPI were added for incubation. Cells were then observed under a confocal microscope, and images were taken. Cell roundness was counted and quantified using ImageJ software. Cytoskeleton staining images (Scale bar, 50 μm) after 3 days of co-culture are shown below. Figure 3 As shown in Figure B, the effect of hydrogel viscoelasticity on cell morphology is illustrated. Quantitative analysis of nucleus pulposus cell roundness based on cytoskeleton staining in the co-culture system is shown below. Figure 3 As shown in Figure C, the slow stress relaxation gel makes the cell roundness closer to 1. Compared with the cells on the surface of the SLOW gel group, the cell roundness in the MEDIUM and FAST groups is significantly reduced, and more cells exhibit an elongated spindle shape.

[0086] To detect the viability of nucleus pulposus cells in three groups of hydrogel matrices, a live / dead cell assay was performed. On the third day after co-culture, LIVE / DEAD staining reagent was added to the wells and incubated at 37°C in the dark for at least 30 minutes, followed by observation using a fluorescence microscope.

[0087] For the EdU cell proliferation experiment, on the third day after co-culture, the culture medium in the 24-well plate was removed, and the cells were washed three times with PBS. EdU staining solution was then added, and the cells were incubated at 37°C for 2 hours. After EdU cell labeling was completed, the supernatant in the 24-well plate was removed, and 400 μL of 4% paraformaldehyde solution was added for fixation. After 15 minutes, the cell fixative was removed, and the cells were washed with PBS at least three times, with each wash lasting 3-5 minutes. Cell permeabilization was then performed by adding immunofluorescence staining permeabilization solution for 15 minutes, followed by washing with PBS twice, and then staining with DAPI for 10 minutes. After removing the staining solution and washing with PBS twice, the cells were observed using a fluorescence microscope and photographed.

[0088] Images of live / dead cell staining and EdU cell proliferation assay after 3 days of co-culture (Scale bars, 400 μm and 100 μm) are shown below. Figure 3 As shown in Figure D, the quantitative analysis of EdU cell proliferation assay is as follows: Figure 3 As shown in Figure E, the EdU positivity rate in the FAST gel group was 33.68 ± 1.48%, which was significantly higher than that in the SLOW group (16.81 ± 1.42%) and the MEDIUM group (26.64 ± 1.76%), suggesting that cell proliferation was more active in the FAST group.

[0089] For Ki-67 flow cytometry analysis, 600 μL of hydrogel precursor was added to each well of a 6-well plate, followed by UV curing. Nucleus pulposus cells were then seeded and co-cultured for 3 days. Afterward, 500 μL of trypsin was added to each well for digestion, and the co-cultured nucleus pulposus cells were collected by centrifugation. The cells were washed twice with PBS to completely remove residual culture medium, and then perforated with 70% ethanol solution at 4°C for 1 hour. Following perforation, the cells were incubated with Ki-67 antibody for 1 hour. The stained nucleus pulposus cells were then analyzed using flow cytometry and FlowJo 10.0 software. The Ki-67 flow cytometry analysis after 3 days of co-culture is shown below. Figure 3 As shown in Figure F, the quantitative analysis of the Ki-67 positivity rate is as follows: Figure 3 As shown in Figure G, the Ki-67 positivity rate in the FAST group was 17.58±1.39%, which was significantly higher than that in the SLOW and MEIDUM groups. This suggests that the proportion of cells in the FAST gel matrix culture was greater in the proliferative state, meaning that the nucleus pulposus cells in the rapid stress relaxation gel group had a better cell proliferation state.

[0090] Example 5

[0091] This embodiment provides a tissue compatibility test for the differentially viscoelastic hydrogels of Example 2, Comparative Example 1, and Comparative Example 2, including the following:

[0092] The tissue compatibility of differentially viscoelastic hydrogels was verified by constructing a rat subcutaneous implantation model. SD rats weighing 300-350 g were used. Three groups of differentially viscoelastic hydrogels were prepared into uniformly sized and shaped hydrogel blocks in a 4mm × 3mm cylindrical mold for subcutaneous implantation. Rats were anesthetized with 2% pentobarbital (2.5 ml / kg), and the skin on their backs was shaved and disinfected. Two longitudinal incisions were made on both sides to form subcutaneous bags, and the three groups of hydrogel blocks were implanted into the bags. The SD rats were euthanized 7 and 14 days post-operation. Subcutaneous tissue was then harvested for histological staining. Simultaneously, the hearts, livers, spleens, lungs, and kidneys of the SD rats were collected, fixed with 4% paraformaldehyde, and then subjected to histological staining. The tissue compatibility test results of the hydrogels are as follows: Figure 4 As shown, where, Figure 4 Image A shows the H&E images (scale bars, 2 mm and 200 μm) of the implanted hydrogel in rats 7 and 14 days after subcutaneous implantation. Figure 4 Image B shows H&E staining images (Scale bar, 200 μm) of the heart, liver, spleen, lungs, and kidneys of rats in each group after subcutaneous implantation. The experimental results show that no obvious tissue necrosis or inflammation was found in the local area of ​​gel implantation or in the major organs of the rats, demonstrating that all three groups of hydrogels have good tissue compatibility.

[0093] Example 6

[0094] This embodiment provides an experimental study on the mechanism by which viscoelastic matrix regulates cellular mechanotransduction, including the following:

[0095] 1. Metabolomics and transcriptomics analysis

[0096] In this embodiment, three differentially viscoelastic hydrogels were co-cultured with nucleus pulposus cells for 3 days. Total nucleic acids were extracted using TRIzol reagent, and RNA purity was measured and quantified using a NanoDrop 2000 spectrophotometer. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biomedical Technology Co., Ltd. Clean reads were obtained using Trimomtic software, and hisat2 was compared with a reference gene. Quantification of exon fragments per million reads per kilobase was performed using cuff software. GO and KEGG enrichment analyses were performed on differentially expressed transcripts to determine the main biological functions or pathways affected by them. For metabolomics experiments, LC-MS analysis was performed by Shanghai Ouyi Biomedical Technology Co., Ltd. Metabolites with statistically significant p < 0.05 were defined as differentially expressed metabolites, and further KEGG enrichment analysis was conducted based on the differentially expressed metabolites to analyze potential pathways. The results of metabolomics and transcriptomics analyses are as follows: Figure 5 As shown, where Figure 5 Figure A shows a volcano plot illustrating the differential metabolite analysis of nucleus pulposus cells co-cultured in slow and fast stress relaxation matrices. Figure 5 Figure B shows a heatmap of the content of differentially metabolites during glycolysis in the slow stress relaxation matrix and the fast stress relaxation matrix groups. Figure 5 The middle F section represents the KEGG enrichment analysis of differentially metabolized products. Figure 5 China A Figure 5 China B and Figure 5 The results from the middle F study showed differences in the metabolic products of cells in the matrix at different stress relaxation rates; Figure 5 Analysis of the content of C in the middle is fructose-6-phosphate. Figure 5 D represents the content analysis of sodium lactate. Figure 5 E represents the content analysis of pyruvate. Figure 5 C, Figure 5 China D and Figure 5 Quantitative analysis of E in the FAST group showed that the intermediate products of glycolysis, fructose-6-phosphate, pyruvate, and sodium lactate, were significantly increased in the FAST group, suggesting that the level of glycolysis in nucleus pulposus cells in the FAST group was significantly higher than that in the MEDIUM and SLOW groups. Figure 5 The experimental results show that hydrogels with rapidly stress-relaxed matrices can enhance the glycolysis process.

[0097] 2. Seahorse metabolic analysis

[0098] The metabolic levels of co-cultured nucleus pulposus cells were investigated using a hippocampal extracellular flux XFe24 metabolic analyzer. After incubation for 1 h in a CO2-free incubator, the surface nucleus pulposus cells in the co-culture system were digested with trypsin, followed by a 1×10⁻⁶ ppm digestion. −5 Cells were seeded at the specified density onto SeahorseXF-24 plates. To measure ECAR, glucose (10 × 10⁻⁶) was added to the plates. -3 M), oligomycin (1×10) −6 M) and 2-DG (50×10 −3 M) were added sequentially to the probe plate. Glycolysis was assessed by subtracting the maximum ECAR value before glucose injection from the final ECAR value before glucose injection. Glycolytic capacity was assessed by subtracting the maximum ECAR value before glucose injection from the maximum ECAR value after oligomycin injection. Seahorse metabolic analysis results are as follows: Figure 6 As shown, Figure 6 Figure A shows the extracellular acidification rate curves of the hippocampus in the three groups. Figure 6 B represents the quantitative analysis results of the basal glycolysis level in the three groups. Figure 6 C represents the quantitative analysis result of glycolytic capacity. Figure 6The results of quantitative analysis of glycolysis reserve in the middle D are as follows: The results show that the FAST group showed the highest basal glycolysis level in the glycolysis stress test, which was 30.5% and 47.3% higher than that of the MEDIUM and SLOW groups, respectively. After the addition of oligomycin, the FAST group also showed the highest glycolysis capacity and glycolysis reserve. Figure 6 The experimental results show that nucleus pulposus cells in gels with rapidly stress-relaxed matrices exhibit a stronger level of glycolysis.

[0099] 3. Detection of oxidative stress levels in nucleus pulposus cells

[0100] For three groups of nucleus pulposus cells co-cultured with differential viscoelasticity, changes in cell membrane potential were detected using JC-1 reagent after 3 days of co-culture. Following the instructions, cells were washed with PBS, then 3 μM JC-1 reagent was added, and incubated at 37°C for 30 minutes. After washing with PBS, membrane potential was observed under a confocal laser microscope.

[0101] Results of oxidative stress level detection in nucleus pulposus cells as follows Figure 7 As shown. JC-1 stained images (Scale bar, 50 μm) of mitochondrial membrane potential of nucleus pulposus cells co-cultured in hydrogels with three differentially viscoelastic matrices: SLOW, MEDIUM, and FAST. Figure 7 As shown in Figure A, the quantitative analysis results of JC-1 staining are as follows: Figure 7 As shown in Figure B, the results indicate that the hydrogel of the slow stress relaxation matrix mediates mitochondrial dysfunction, leading to early apoptosis of nucleus pulposus cells. Conversely, the mitochondrial function of nucleus pulposus cells in the FAST group was better than that in the MEDIUM and SLOW groups, demonstrating that the hydrogel of the rapid stress relaxation matrix is ​​beneficial to the recovery of mitochondrial membrane potential.

[0102] For observation of mitochondrial microstructure, nucleus pulposus cells co-cultured for 3 days were digested with trypsin, centrifuged, and the precipitate collected at the bottom of EP tubes. After discarding the supernatant, 2.5% glutaraldehyde was slowly added for fixation for 1 hour, followed by fixation in 2% osmium tetroxide for 3 hours. Cells were then stained with 0.5% uranyl acetate for 12 hours. After dehydration and polymerization, the samples were sliced ​​into ultrathin sections of 70-90 nm using a microtome, and mitochondrial morphology of the differentially viscoelastic matrix was assessed by TEM. Figure 7 C represents the mitochondrial morphology observation results of the differential viscoelastic matrix hydrogel group. The results show that the mitochondria of nucleus pulposus cells in the FAST group are oval, and the mitochondrial cristae are arranged more neatly and clearly distributed. Its morphology is better than that of the MEDIUM and SLOW groups, and it has better microscopic morphology.

[0103] The level of intracellular reactive oxygen species (ROS) in cells was detected using the DCFH-DA probe. After co-culturing for 3 days, the suspension cells were digested with trypsin. The cells were thoroughly washed with PBS, and DCFH-DA working solution was added. The cells were incubated at 37°C in the dark for 30 min. The supernatant was centrifuged and washed with PBS for flow cytometry analysis.

[0104] For the detection of cell superoxide, staining was performed using the Mito-SOX RED staining kit. After co-culturing for 3 days, the supernatant of each group was discarded, and the cells were washed with PBS and digested with trypsin. After centrifugation, 500 μL of Mito-SOX RED working solution was added to each 6-well plate, and the cells were incubated at 37°C for 60 minutes. After centrifugation at 600×g at 4°C for 3-4 minutes, the cells were resuspended in 1 mL of PBS and analyzed by flow cytometry. The results of DCFH-DA analysis of nucleus pulposus cells after co-culturing with a hydrogel containing a differentially viscoelastic matrix are shown below. Figure 7 As described in D, the results of MitoSOX flow cytometry analysis are as follows: Figure 7 As shown in Figure E, the results indicate that the FAST gel matrix can restore mitochondrial membrane potential by reducing ROS accumulation, demonstrating that the level of oxidative stress damage in hydrogels with rapid stress relaxation matrix is ​​significantly improved compared to those with slow stress relaxation.

[0105] 4. Detection of apoptosis levels

[0106] For Annexin V-FITC apoptosis detection, after co-culturing for 3 days, cells were digested with trypsin, centrifuged at 1000g for 5 minutes, and the cells were collected and counted. 200μL of prepared Annexin V-FITC working solution was added first, followed by 10μL of PI staining solution. The cells were gently pipetted and resuspended, and incubated at room temperature in the dark for 20 minutes. The cells were then detected by flow cytometry.

[0107] The results of the detection of apoptosis level are as follows: Figure 8 As shown. Figure 8 Image A shows the results of Annexin V-FITC / PI assay for apoptosis levels in nucleus pulposus cells. The results indicate that as the rate of stromal stress relaxation gradually increases, Annexin V-FITC... + / PI + The proportion of late-stage apoptotic cells gradually decreased, suggesting that in the rapid stress relaxation gel group, the apoptosis process was alleviated as cellular metabolic disorders were corrected and ROS was reduced.

[0108] For TUNEL staining, a one-step TUNEL apoptosis detection kit was used. After co-culturing for 3 days, the culture medium was discarded, and the cells were washed with PBS, fixed with 4% paraformaldehyde, washed once with PBS, and permeabilized with immunofluorescence permeabilization buffer. Then, 50 μL of TUNEL working staining solution was added to each 24-well plate, and incubated at 37°C for 60 minutes. During incubation, the wells were sealed with sealing film to prevent evaporation. After staining, the cells were washed three times with PBS, followed by staining with DPAI for 10 minutes. After washing, the cells were observed under a fluorescence microscope. Figure 8 Image B shows the experimental results of hydrogel TUNEL staining with three different stress relaxation rates. Figure 8 C represents the quantitative analysis results of TUNEL staining, indicating that TUNEL... + The proportion of cells decreased significantly in the FAST group, demonstrating a lower proportion of apoptosis in the rapid stress relaxation gel.

[0109] The above examples demonstrate that the viscoelastic hydrogels of the three stress relaxation matrices have similar Young's moduli and good cell and tissue compatibility. However, the cell roundness in the FAST group was significantly reduced, with more cells exhibiting an elongated spindle shape. The nucleus pulposus cells in the rapid stress relaxation gel group showed better cell proliferation, indicating that the co-culture system provided more suitable ECM components or growth signals. The hydrogels of the rapid stress relaxation matrix can enhance the glycolysis process, exhibiting a stronger level of glycolysis, which is beneficial for the recovery of mitochondrial membrane potential. The level of oxidative stress damage in the rapid stress relaxation matrix hydrogel was significantly improved compared to the slow stress relaxation gel, and the proportion of apoptosis was lower. These experimental results demonstrate that the hydrogels of the rapid stress relaxation matrix can promote cell growth, proliferation, and migration through viscoelastic mechanical properties, thereby promoting tissue repair.

[0110] Example 7

[0111] This embodiment provides a method for preparing viscoelastic dual-network hydrogel microspheres, including the following steps:

[0112] Viscoelastic hydrogel microspheres were prepared using a water-in-oil microfluidic method:

[0113] Microfluidic chips with a channel width of 100 μm were used for fabrication. The microfluidic chip was connected to a microfluidic pusher via a silicone tube. A 5 ml syringe placed on the pusher contained a 2% AHAMA / COL precursor solution as the continuous phase, while another 5 ml syringe connected to a silicone tube contained a 10% (w / w) Span 80 isopropyl myristate solution as the shear phase. The flow rates of the shear phase and continuous phase in the microfluidic device were adjusted to 40 μL / min and 2 μL / min, respectively, to form continuous monodisperse spherical droplets at the interface of the main phase. Once the droplets were uniform in size, they were placed in a beaker containing the shear phase under light-protected conditions to collect the microspheres. The beaker was removed every 10–20 minutes and thoroughly irradiated with ultraviolet light while being stirred at 200 rpm to prevent the prepared microsphere droplets from sticking together. The collected cross-linked hydrogel microspheres were washed three times with 75% ethanol and five times with PBS. The PBS was then replaced every 3 hours to remove the photoinitiator and residual shear phase on the surface of the microspheres. The purified microspheres were frozen at -80°C overnight, and then immediately freeze-dried to obtain porous hydrogel microspheres with a rapid stress relaxation matrix, which were named FMS. The morphology of the porous hydrogel microspheres was observed under SEM according to the method in Example 3.

[0114] Comparative Example 3

[0115] This comparative example provides a method for preparing viscoelastic dual-network hydrogel microspheres. Under the same conditions as in Example 7, the 2% AHAMA / COL precursor solution is replaced with 2% HAMA / COL to obtain porous hydrogel microspheres with a slow stress relaxation matrix, which are named SMS.

[0116] Comparative Example 4

[0117] This comparative example provides a method for preparing viscoelastic dual-network hydrogel microspheres. Under the same conditions as in Example 7, the 2% AHAMA / COL precursor solution was replaced with 1% HAMA / 1% AHAMA / COL to obtain porous hydrogel microspheres with a medium-speed stress relaxation matrix, which were named MMS. The characterization and analysis results of the three types of hydrogel microspheres are as follows: Figure 9 As shown, where, Figure 9 Figure A shows the microstructure of FMS, SMS, and MMS observed under SEM. The results indicate that, similar to the microstructure of bulk hydrogels, there are no significant differences in the microstructure of FMS, SMS, and MMS.

[0118] Example 8

[0119] This embodiment provides a method for preparing cell-loaded microspheres of differentially viscoelastic dual-network hydrogel microspheres according to Example 7, Comparative Example 3, and Comparative Example 4, including the following steps:

[0120] In Example 7, after the differentially viscoelastic porous hydrogel microspheres of Comparative Examples 3 and 4 were prepared, they were soaked overnight in 75% ethanol. The next day, the microspheres were washed with PBS. Then, the FMS, SMS, and MMS microspheres were placed at the bottom of a 24-well anti-adhesion plate (eFL-TCP-0024) from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., and subsequently seeded with nucleus pulposus cells. The microspheres prepared with the hydrogel at a rapid stress relaxation rate were named FMS-NP, those prepared with the hydrogel at a medium stress relaxation rate were named MMS-NP, and those prepared with the hydrogel at a slow stress relaxation rate were named SMS-NP. The characterization and analysis results of the three types of hydrogel microspheres seeded with nucleus pulposus cells are as follows: Figure 9 As shown, where, Figure 9 Image B shows the microstructure of three different viscoelastic hydrogel microspheres observed under SEM. FMS-NP, SMS-NP, and MMS-NP cell-carrying microspheres were directly observed under an optical microscope, and their cytoskeleton structure was subsequently observed using immunofluorescence staining. The immunofluorescence staining results are shown below. Figure 9 As shown in Figure B, the results indicate that after 7 days of co-culture, the surface of the microspheres was covered with nucleus pulposus cells, demonstrating excellent biocompatibility of the three types of hydrogels. Simultaneously, the nucleus pulposus cells exhibited good proliferation at the junctions between the microspheres. Immunofluorescence staining of proteoglycans and type II collagen was performed on FMS-NP, SMS-NP, and MMS-NP. Figure 9 Image C shows the immunofluorescence images of type II collagen and proteoglycan in three types of cell-carrying hydrogel microspheres. Figure 9 Figure D shows the results of quantitative analysis of immunofluorescence images. The results show that, similar to nucleus pulposus cells co-cultured directly on the surface of bulk hydrogels, the nucleus pulposus cells in the rapid stress relaxation gel exhibit a stretched spindle shape with a larger cell aspect ratio. Quantitative analysis revealed that the fluorescence intensity of Aggrecan and COL-II in the FMS-NP group was significantly higher than that in the MMS-NP and SMS-NP groups. The results demonstrate that the function of the rapid stress relaxation cell-carrying hydrogel microspheres is similar to that of the bulk hydrogel, and it can significantly enhance ECM secretion.

[0121] Example 9

[0122] This embodiment provides a rat model of IVDD (infectious viscoelastic dual-network hydrogel microspheres) established using Example 7, Comparative Example 3, and Comparative Example 4, and evaluates the effectiveness of the differentially viscoelastic dual-network hydrogel microspheres, including the following:

[0123] 1. Establish an IVDD rat acupuncture model to evaluate the therapeutic effect of targeted hydrogel.

[0124] First, animals were anesthetized with 2% sodium pentobarbital at a concentration of 2.5 ml / kg via intraperitoneal injection. After complete anesthesia, the Co7-8 and Co8-9 intervertebral discs were punctured using a 21-gauge needle. When the center of the intervertebral disc was punctured (puncture depth 5 mm), the needle was rotated for 5 seconds and then held still for 30 seconds. After the puncture was completed, 20 μL of PBS, SMS, MMS, and FMS microsphere solutions were injected, respectively. Animals that did not undergo surgery were designated as Control; animals that received PBS after puncture were designated as PBS group; animals that received SMS-NP after puncture were designated as SMS-NP group; animals that received MMS-NP after puncture were designated as MMS-NP group; and animals that received FMS-NP after puncture were designated as FMS-NP group. To assess the degradation rate of hydrogel microspheres in the intervertebral disc, Cy5-loaded hydrogel microspheres of TargetMol+T15025L were prepared and injected into the Co7-8 segment. Fluorescence intensity was measured and analyzed at 0, 3, 5, 7, and 14 days post-surgery using an in vivo small animal imaging system (IVIS Lumina Xrms). The small animal imaging results are as follows: Figure 10 As shown. Figure 10 Image A shows small animal imaging of three types of cell-carrying hydrogel microspheres in the rat tailbone environment. Figure 10 The fluorescence intensity of small animal imaging images at 0, 3, 5, 7, and 14 days was quantitatively analyzed. The results showed that the overall degradation rate of the three groups of hydrogel microspheres was similar. From the 3rd day after injection, the fluorescence intensity of all three groups of hydrogel microspheres decreased significantly, and by the 14th day, the fluorescence intensity of all three groups was almost gone. This shows that the three types of hydrogel microspheres have similar retention efficiency in vivo, proving that the dual-network hydrogel microspheres can remain in vivo for about 2 weeks, which provides a time window for the viscoelastic matrix to promote the repair of nucleus pulposus cells.

[0125] 2. Radiographic evaluation of animal specimens

[0126] Rats underwent X-ray and MRI scans at 4 and 8 weeks post-surgery to observe changes in intervertebral disc height and signal intensity. The disc height index (DHI) was calculated using X-ray imaging, and the ratio of DHI in the experimental group to that in the control group was recorded as DHI%. Each intervertebral disc was graded based on T2-weighted MRI results, referencing a modified Thomson grading system. The imaging evaluation results of the animal specimens are as follows: Figure 11 As shown, where, Figure 11 Image A shows X-ray and T2-weighted MRI images of the rat caudal vertebrae within each group at weeks 4 and 8 after the animal model was established. Figure 11 In section B, a quantitative analysis of the changes in intervertebral disc height index (DHI%) was performed on the five groups at 4 and 8 weeks. Figure 11C represents the quantitative analysis of MRI grading at 4 weeks based on T2-weighted MRI images. Figure 11 The data in the middle section represents the quantitative analysis of MRI grading at 8 weeks using T2-weighted MRI images. The results showed that intervertebral disc height decreased significantly in all groups after puncture, with a more pronounced decrease at 8 weeks compared to 4 weeks. However, the rapid stress relaxation microsphere group showed the smallest decrease, more closely resembling the non-puncture Control group. In contrast, the DHI index in the slow stress relaxation gel group was closer to that of the PBS injection group, demonstrating the in vivo repair effect of the rapid stress relaxation matrix. MRI scoring results showed that a significant increase in MRI index occurred in all puncture groups. However, after injection of the three types of cell-loaded hydrogel microspheres, there was some signal recovery in T2 imaging, proving that the introduction of collagen components and the nucleus pulposus cell transplantation strategy had a certain effect in vivo. However, only the FMS-NP group had the closest MRI signal in T2 imaging to the non-puncture Control group. The slow and medium-speed stress relaxation cell-loaded microsphere groups had limited repair effects in vivo because they did not provide suitable viscoelastic mechanical cues. This indicates that the rapid stress relaxation cell-loaded microsphere FMS-NP has the function of restoring the height of the rat caudal intervertebral disc and restoring the grading under MRI.

[0127] 3. Histological evaluation of animal specimens

[0128] After euthanasia of rats due to overdose of anesthesia, intervertebral disc tissue was removed and fixed with 4% paraformaldehyde, followed by decalcification in 10% EDTA solution. After embedding in paraffin blocks, the tissue specimens were cut into 5μm sections, including the endplate, nucleus pulposus, and annulus fibrosus. Histological evaluation of the intervertebral discs in different groups was performed after H&E and S&O staining. The histological evaluation results of the animal specimens are as follows: Figure 12 As shown, Figure 12 Image A shows tissue H&E images and red-to-green images for each group at 4 and 8 weeks. Figure 12 Figure B shows the histological grading and quantitative analysis performed on tissue sections at 4 and 8 weeks after puncture. The results showed that after puncture and injection of PBS, no nucleus pulposus tissue was observed at 4 and 8 weeks, and it was completely replaced by regenerated scar tissue. However, after injection of cell-loaded hydrogel microspheres, the nucleus pulposus tissue underwent a certain degree of regeneration. In the FMS-NP group, the boundary between the nucleus pulposus and the annulus fibrosus tissue became more obvious, the nucleus pulposus volume increased significantly, and its histological score was closer to that of the Control group, indicating that the rapid stress relaxation cell-loaded microspheres FMS-NP have better in vivo tissue repair efficacy.

[0129] The above embodiments, through the construction of clinical tissue specimens and animal models, revealed the pathological relationship between the synchronous downregulation of viscoelasticity in the nucleus pulposus and the expression of multiple glycolysis-related enzymes as intervertebral disc degeneration progresses. This provides a foundation for in-depth research into the regulatory mechanism of matrix viscoelasticity on NPC fate. By adjusting the content of dynamic imine bonds in the aldehyde-modified hyaluronic acid-collagen dual network, and based on the successful construction of a two-dimensional cell culture platform with tunable viscoelasticity independent of elastic modulus, the embodiments integrated metabolomics and transcriptomics to systematically analyze and verify the improvement in cellular glycolysis levels brought about by rapidly relaxed matrix, demonstrating the potential of viscoelasticity in the preparation of drugs for tissue repair and treatment of intervertebral disc degeneration.

Claims

1. Application of a viscoelastic dual-network hydrogel in the preparation of drugs that promote tissue repair.

2. The application according to claim 1, characterized in that, The tissue in question is the nucleus pulposus.

3. The application according to claim 1, characterized in that, The viscoelastic dual-network hydrogel promotes tissue repair by enhancing cellular glycolysis through a viscoelastic matrix.

4. The application according to claim 1, characterized in that, The viscoelastic dual-network hydrogel promotes tissue repair by increasing the extracellular matrix content in tissues through a viscoelastic matrix.

5. The application according to claim 1, characterized in that, The viscoelastic dual-network hydrogel is a porous hydrogel with a rapid stress relaxation matrix.

6. The application according to claim 5, characterized in that, The preparation of the viscoelastic dual-network hydrogel includes the following steps: Sodium periodate was added to a hyaluronic acid solution and stirred under dark conditions to modify it. The modified solution was then post-treated to obtain aldehyde-modified hyaluronic acid. Aldehyaluronic acid was dissolved, methacrylate was added to the aldehyde-modified hyaluronic acid solution, the pH of the solution was adjusted to 8-8.5, the pH-adjusted solution was placed in an ice bath to carry out the substitution reaction, and the substitution reaction product was post-treated to obtain aldehyde- and methacryloyl-modified hyaluronic acid. Aldehyde and methacryloyl-modified hyaluronic acid were dissolved in a photoinitiator solution, and bovine collagen powder was added to the dissolved solution to obtain a hydrogel precursor solution. Bulk viscoelastic dual-network hydrogels or viscoelastic dual-network hydrogel microspheres were prepared using hydrogel precursor solutions.

7. The application according to claim 6, characterized in that, The steps for preparing bulk viscoelastic dual-network hydrogels from hydrogel precursor solutions include: placing the hydrogel precursor solution under 365 nm ultraviolet light irradiation to obtain bulk viscoelastic dual-network hydrogels.

8. The application according to claim 6, characterized in that, The steps for preparing viscoelastic dual-network hydrogel microspheres from hydrogel precursor solutions include: preparing microspheres from hydrogel precursor solutions using a water-in-oil microfluidic method, and post-processing the microspheres to obtain viscoelastic dual-network hydrogel microspheres.

9. The application according to claim 6, characterized in that, The mass ratio of photoinitiator to aldehyde and methacryloyl-modified hyaluronic acid is 1:

8.

10. Application of a viscoelastic dual-network hydrogel in the preparation of therapeutic drugs for intervertebral disc degeneration.

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