Viscoelastic hydrogels with tunable viscosity and their use in assessing the effect of hydrogel viscosity on cell behavior

Abstract

The application belongs to the field of medical modified and natural polymer hydrogel, and provides a viscoelastic hydrogel with adjustable viscosity and application thereof in evaluation of influence of hydrogel viscosity on cell behavior. Through a double crosslinking mechanism of covalent crosslinking and ionic crosslinking, two viscoelastic hydrogels with the same initial modulus but different viscosities are prepared. The covalent crosslinking hydrogel is composed of methacrylated alginate (AlgMA), gelatin (Gel) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and the ionic crosslinking hydrogel is composed of sodium alginate (Alg), gelatin (Gel) and calcium chloride. The application realizes fine adjustment of the viscosity of the hydrogel without changing the initial modulus by precisely controlling the crosslinking degree, which has an important influence on neural stem cell adhesion and survival. The experimental method provided by the application verifies the key role of the viscosity of the viscoelastic matrix in the regulation of cell behavior.

Description

Technical Field

[0001] This invention belongs to the field of medical modified and natural polymer hydrogels, and relates to a method for preparing viscoelastic hydrogels with adjustable viscosity and an experimental verification method for regulating the behavior of neural stem cells by viscosity. The hydrogel is suitable for regulating the adhesion of neural stem cells and can be used for tissue repair, drug screening, etc. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With advancements in science and technology, bio-hydrogel preparation technology and 3D printing technology have provided novel clinical medical strategies for tissue and organ repair. Applications in the medical field include the construction of tissue and organ replacements, organ transplantation and regeneration, drug screening models, and anatomical models. Suitable physicochemical properties of hydrogel materials are prerequisites for cell adhesion, migration, and proliferation. Therefore, hydrogel materials must possess good biocompatibility, providing a specific microenvironment for cell growth and serving as the basis for inducing cell functional behavior.

[0004] Gelatin, derived from the denaturation of collagen, is a versatile biomaterial with broad application prospects in cell culture, tissue engineering, and 3D printing. One of the outstanding characteristics of gelatin hydrogels is their ability to serve as an excellent matrix for cell culture and tissue growth. Similar to collagen, a fundamental component of the extracellular matrix, they contain numerous bioactive sites, particularly the integrin-binding sequence RGD peptide, exhibiting both good biodegradability and the ability to regulate cell adhesion. This biocompatibility is essential in tissue engineering, and gelatin hydrogels provide a nurturing environment for developing functional tissue structures.

[0005] Sodium alginate, primarily derived from abundant brown seaweed, is widely used as a base material for preparing hydrogels. Its popularity is largely due to its remarkable characteristics, including biocompatibility, adaptive mechanical properties, and rapid gelation behavior. First, sodium alginate hydrogels exhibit excellent biocompatibility, making them a preferred choice for a wide range of biomedical applications. This biocompatibility ensures that alginate-based constructs can interact seamlessly with living tissues and biological systems, minimizing the risk of adverse or immune responses; therefore, they are ideally suited for regenerative medicine, tissue engineering, and drug delivery. Another significant feature of sodium alginate hydrogels is their tunable mechanical properties. These hydrogels provide engineers and researchers with significant control over their mechanical properties. Furthermore, alginate hydrogels are known for their rapid gelation in the presence of calcium ions; simultaneously, alginate can be modified to achieve photocrosslinking.

[0006] Many past studies investigating the effects of mechanical properties on cell behavior have neglected the viscoelastic behavior of hydrogel matrices and various tissues, instead using hydrogels that exhibit purely elastic behavior. Tissues such as muscle are viscoelastic. Therefore, if viscoelasticity, particularly the viscosity of hydrogels, is incorporated into the design of the hydrogel matrix, it can modulate cell behavior. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides viscoelastic hydrogels with adjustable mechanical properties and viscosity based on different crosslinking methods of sodium alginate, and provides an experimental verification method for the regulation of neural stem cell behavior by viscosity. This invention prepares viscoelastic hydrogels with good biocompatibility, adjustable viscosity, and rapid gelation, and proposes an experimental verification method for the regulation of cell behavior by viscoelastic hydrogel viscosity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides a method for preparing viscoelastic hydrogels with the same initial modulus but different viscosities, comprising:

[0010] Covalently cross-linked sodium alginate-based viscoelastic hydrogels and ionically cross-linked sodium alginate-based viscoelastic hydrogels were prepared respectively.

[0011] Among them, multiple sets of covalently cross-linked viscoelastic hydrogels were obtained according to different polymerization times;

[0012] Based on different ion crosslinking concentrations, multiple sets of ion-crosslinked viscoelastic hydrogels were obtained;

[0013] The initial elastic modulus of multiple covalently cross-linked viscoelastic hydrogels and ionicly cross-linked viscoelastic hydrogels were measured. Covalently cross-linked viscoelastic hydrogels and ionicly cross-linked viscoelastic hydrogels with similar initial elastic modulus were grouped together, and the viscosity of this group of hydrogels was tested. If the two viscoelastic hydrogels had different viscoelastic moduli, viscoelastic hydrogels with the same initial modulus but different viscoelastic ...

[0014] Currently, any polymeric material, such as gelatin, polyvinyl alcohol (PVA), and hyaluronic acid (HA), can form hydrogels through different cross-linking mechanisms (such as physical and chemical cross-linking), and their initial moduli can be adjusted to be the same. However, the matrix composition of any two hydrogels cannot be guaranteed to be the same, meaning that the influence of the matrix composition on cell behavior cannot be ruled out. Therefore, this invention, for the first time, leverages the unique property of sodium alginate, which can achieve both ionic cross-linking and covalent cross-linking through modification, to form two different hydrogel networks without changing the hydrogel matrix composition. By adjusting the degree of cross-linking of the two hydrogels, this invention studies how different cross-linking mechanisms lead to different viscosities, thereby affecting cell behavior, while ensuring that the hydrogel composition and matrix modulus (stiffness) are the same.

[0015] In some embodiments, the method for preparing the covalently crosslinked viscoelastic hydrogel includes:

[0016] Methacrylated alginate, gelatin, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate were mixed evenly in a solvent to obtain a mixed solution;

[0017] The mixed solution was polymerized under ultraviolet light to obtain a covalently cross-linked viscoelastic hydrogel.

[0018] In some embodiments, the mass ratio of the methacryloyl alginate, gelatin, and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate is 2:0.5:0.25-0.3.

[0019] In some embodiments, the preparation method of the ion-crosslinked viscoelastic hydrogel includes:

[0020] Prepare a serum-free DMEM solution containing sodium alginate and gelatin;

[0021] Prepare a serum-free DMEM solution containing calcium chloride;

[0022] A serum-free DMEM solution containing calcium chloride and a serum-free DMEM solution containing sodium alginate and gelatin were mixed, and the resulting sediment was collected to obtain an ion-crosslinked viscoelastic hydrogel.

[0023] In some embodiments, the mass ratio of sodium alginate to gelatin is 2:0.5.

[0024] In some embodiments, the concentration of calcium chloride is 24–80 mM.

[0025] A second aspect of the invention provides the application of the aforementioned viscoelastic hydrogels with the same initial modulus but different viscosities in evaluating the effect of hydrogel viscosity on cell behavior.

[0026] In some embodiments, the cells are neural stem cells.

[0027] In some implementations, the cell behavior refers to cell adhesion and cell survival.

[0028] A third aspect of the present invention provides a method for evaluating the effect of viscoelastic hydrogel viscosity on the adhesion and survival of neural stem cells, comprising:

[0029] At least one group of viscoelastic hydrogels with the same initial modulus but different viscosities were cured on a perforated plate, allowed to stand, washed, and the perforated plate with different cured hydrogels was obtained.

[0030] Neural stem cells were seeded onto well plates with different solidified hydrogels, cultured, and the cell adhesion area and the number of live and dead cells were statistically analyzed. The effect of viscoelastic hydrogel viscosity on the adhesion and survival of neural stem cells was evaluated based on the statistical results.

[0031] Beneficial effects of the present invention

[0032] (1) This invention prepares viscoelastic hydrogels with the same initial modulus but different viscosities through two crosslinking mechanisms. The viscoelastic hydrogels have the characteristics of adjustable viscosity, good biocompatibility and rapid gelation, and provide an experimental method to verify the effect of viscoelastic hydrogel viscosity on cell behavior.

[0033] (2) This invention, for the first time, leverages the unique property of sodium alginate, which can achieve both ionic crosslinking and covalent crosslinking through modification, to form two different hydrogel networks without altering the composition of the hydrogel matrix. By adjusting the degree of crosslinking in the two hydrogels, this invention investigates how different crosslinking mechanisms lead to different viscosities and thus affect cell behavior, while ensuring the same hydrogel composition and matrix modulus (stiffness). The preparation method of this invention is simple, practical, and easy to promote. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 (a) and (b) show the chemical structures of the covalently cross-linked and ionicly cross-linked sodium alginate hydrogels of the present invention, respectively.

[0036] Figure 2 Infrared spectra of covalently cross-linked viscoelastic hydrogels, ionicly cross-linked viscoelastic hydrogels, alginate, and sodium alginate with methacrylamide. 1-Photocurable covalently cross-linked hydrogel, 2-ionicly cross-linked hydrogel, 3-sodium alginate, 4-methacrylamide sodium alginate.

[0037] Figure 3 This invention illustrates the effect of photocrosslinking time on the storage modulus of covalently crosslinked hydrogels in an embodiment of the present invention.

[0038] Figure 4 For the embodiments of the present invention, Ca 2+ Effect of concentration on the storage modulus of ion-crosslinked hydrogels.

[0039] Figure 5 To select the initial elastic modulus of three groups of hydrogels with comparable initial elastic modulus due to covalent crosslinking and ionic crosslinking mechanisms.

[0040] Figure 6(a) Normalized stress relaxation curves of hydrogels with covalent and ionic crosslinking mechanisms. (b) Storage modulus and loss modulus of ionic and covalent crosslinked hydrogels as a function of frequency.

[0041] Figure 7 The viscosity of viscoelastic hydrogels with two crosslinking mechanisms having comparable initial modulus.

[0042] Figure 8 (a) Effect of viscoelastic hydrogel viscosity on cell adhesion area. (b) Statistics on the adhesion area of ​​neural stem cells NE-4C on viscoelastic matrices with different viscoelasticities under the same initial modulus.

[0043] Figure 9 (a) Representative images of live and dead staining of NE-4C neural stem cells on viscoelastic matrices with low and high viscosity at an initial modulus of 4 kPa. (b) Viability of NE-4C neural stem cells on viscoelastic matrices with different viscoelastic ... Detailed Implementation

[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] The covalently cross-linked viscoelastic hydrogel provided in this embodiment of the invention is composed of the following substances: 2% w / v methacrylamide alginate (AlgMA), 0.5% w / v gelatin (Gel) (providing cell adhesion binding sites), and 0.25% w / v lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP); the ionically cross-linked viscoelastic hydrogel provided in this embodiment of the invention is composed of the following substances: 2% w / v sodium alginate (Alg), 0.5% w / v gelatin (Gel) (providing cell adhesion binding sites), and 24-80 mM calcium chloride.

[0046] The high-viscosity viscoelastic hydrogel provided in this invention significantly promotes the adhesion and survival of neural stem cells, offering broad application prospects for tissue regeneration and tissue repair.

[0047] Embodiments of the present invention provide methods for preparing low-viscosity viscoelastic hydrogel AlgMA-Gel and high-viscosity viscoelastic hydrogel Alg-Gel, the preparation methods comprising:

[0048] A method for preparing viscoelastic hydrogels AlgMA-Gel via covalent crosslinking mechanism

[0049] Step 1: Prepare a mixed solution containing 2% w / v of methacrylamide alginate (AlgMA), 0.5% w / v of gelatin, and 0.25% w / v of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP).

[0050] Step 2: Irradiate the mixed solution prepared in Step 1 with 405nm ultraviolet light for 60-600s to prepare hydrogels with different initial elastic moduli.

[0051] A method for preparing viscoelastic hydrogels Alg-Gel via ionic crosslinking

[0052] Step 3: Prepare a serum-free DMEM solution containing 2% w / v sodium alginate (100kDa) and 0.5% w / v gelatin.

[0053] Step 4: Prepare serum-free DMEM solutions containing 24mM, 36mM, 56mM, and 80mM calcium chloride solutions, respectively.

[0054] Step 5: Serum-free DMEM solutions of 24mM, 36mM, 56mM, and 80mM calcium chloride solutions, and 2% w / v sodium alginate and 0.5% w / v gelatin solutions were mixed at a volume ratio of 1:4 in a 5ml syringe using a Luer lock at room temperature and rapidly deposited into a custom mold to obtain viscoelastic hydrogels with different ionic crosslinking concentrations.

[0055] An experimental method for obtaining viscoelastic hydrogels with the same initial modulus but different viscosities through two different crosslinking mechanisms:

[0056] Step 6: The chemical structures and cross-linking mechanisms of the two proposed hydrogels were chemically verified and confirmed by Fourier transform infrared spectroscopy.

[0057] Step 7: Verify through rheological experiments that hydrogels with different moduli can be obtained by changing the exposure time (covalent crosslinked hydrogel) and the ionic crosslinking concentration (ionic crosslinked hydrogel).

[0058] Step 8: The initial elastic modulus of hydrogels with different light exposure times (covalent crosslinking) and ionic crosslinking concentrations were measured using a single-column mechanical testing machine. In the elastic modulus test, the gel was compressed to 20% strain at a deformation rate of 1 mm / min, and the slope of the linear stress-strain curve between 10% and 15% strain was obtained.

[0059] Step 9: Based on the experimental results of Step 8, select three groups of hydrogels with comparable initial elastic moduli (covalently cross-linked hydrogels and ionicly cross-linked hydrogels with comparable initial elastic moduli), with initial elastic moduli of low, medium and high, respectively.

[0060] Step 10: Test stress relaxation of covalently cross-linked and ionicly cross-linked hydrogels with equivalent initial elastic modulus. In the stress relaxation measurement, the gel is compressed to 15% strain at a deformation rate of 1 mm / min, thus keeping the strain constant, and then the stress change over time is recorded.

[0061] Step 11: Testing the initial elastic modulus equivalent to viscosity tests of covalently cross-linked and ionicly cross-linked hydrogels. The storage modulus G′ of the cured hydrogel as a function of time and the viscosity of the hydrogel were tested at a fixed frequency (f = 1 Hz) and strain (γ = 1%).

[0062] Experimental methods for regulating neural stem cell behavior by viscoelastic hydrogel viscosity

[0063] Step 12: Verification of the effect of viscoelastic hydrogel viscosity on neural stem cell adhesion.

[0064] Step 12-1: Apply a quantitative amount (0.2 ml / well) of three groups of hydrogels with similar initial moduli and different viscosities (low, medium and high elastic moduli respectively) to a 24-well plate, cure them according to the methods in Step 2 and Step 5, and let them stand for 2 hours.

[0065] Step 12-2: Wash the hydrogel prepared in step 10-1 twice with serum-free culture medium containing 1% penicillin / streptomycin and equilibrate overnight.

[0066] Step 12-3: The cultured neural stem cells NE-4C are then subjected to a 10... 4 pcs / cm 2 Cells were seeded at a density of [missing information] into 24-well plates and cultured for 24 hours. Then, Action-Tracker Green was diluted 1:40 with PBS containing 1% BSA and 0.1% Trion X-100 and stained for 45 minutes. Cells were then stained with DAPI working solution (10 μg / ml) for 10 minutes. During this period, cells were washed multiple times with PBS containing 0.1% Trion X-100 before and after staining. The stained cells were then imaged under a 40x objective lens using an inverted fluorescence microscope, and the cell adhesion area was analyzed using Image software.

[0067] Step 13: Experimental verification of the effect of viscoelastic hydrogel viscosity on neural stem cell survival

[0068] Step 13-1: Apply a quantitative amount (0.2 ml / well) of three groups of hydrogels with similar initial moduli and different viscosities (low, medium and high elastic moduli respectively) to a 24-well plate, cure them according to the methods in Step 2 and Step 5, and let them stand for 2 hours.

[0069] Step 13-2: Wash the hydrogel prepared in step 10-1 twice with serum-free culture medium containing 1% penicillin / streptomycin and equilibrate overnight.

[0070] Step 13-3: NE-4C neural stem cells (2 × 10^4 cells per square centimeter) were seeded in 24-well plates with different solidified hydrogels and cultured at 37°C and 5% CO2 for 48 hours. Dead and live cells were stained with PI and Calcein AM, respectively. The stained cells were then imaged under a 10x objective lens using an inverted fluorescence microscope. After imaging, the number of live and dead cells was statistically analyzed using ImageJ software.

[0071] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0072] Example 1

[0073] The embodiments of the present invention provide two types of hydrogel-covalently cross-linked low-viscosity viscoelastic hydrogels, AlgMA-Gel and ionically cross-linked high-viscosity viscoelastic hydrogels, and their preparation methods. These methods can yield two types of viscoelastic hydrogels with the same initial elastic modulus but different viscosities. Furthermore, the present invention provides an experimental verification method for the regulation of neural stem cell behavior by the viscosity of viscoelastic hydrogels.

[0074] The covalently cross-linked low-viscosity viscoelastic hydrogel AlgMA-Gel provided in this embodiment of the invention is composed of the following substances: 2% w / v methacryloyl alginate (AlgMA), 0.5% w / v gelatin (providing cell adhesion binding sites), and 0.25% w / v lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP); the ionically cross-linked high-viscosity viscoelastic hydrogel Alg-Gel provided in this embodiment of the invention is composed of the following substances: 2% w / v sodium alginate, 0.5% w / v gelatin (Gel) (providing cell adhesion binding sites), and 24-80 mM calcium chloride.

[0075] The high-viscosity viscoelastic hydrogel provided in this invention significantly promotes cell adhesion, offering broad application prospects for tissue regeneration and repair.

[0076] Embodiments of the present invention provide a method for preparing covalently cross-linked low-viscosity viscoelastic hydrogel AlgMA-Gel and ionically cross-linked high-viscosity viscoelastic hydrogel Alg-Gel. The preparation method includes:

[0077] A method for preparing low-viscosity viscoelastic hydrogels AlgMA-Gel via covalent crosslinking.

[0078] Step 1: Prepare a mixed solution containing 2% w / v of methacrylamide alginate (AlgMA), 0.5% w / v of gelatin, and 0.25% w / v of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP).

[0079] Step 2: Irradiate the mixed solution prepared in Step 1 with 405 nm ultraviolet light for 60–600 s to prepare hydrogels with different initial elastic moduli. The chemical structure of the sodium alginate covalently crosslinked hydrogel is as follows: Figure 1 As shown in (a).

[0080] Step 1 of the above scheme is specifically as follows: Measure 10 ml of ultrapure water, add 0.025 g of photoinitiator LAP, and heat in a 40-50℃ water bath for 15 min to dissolve, stirring and shaking several times during the process to obtain a 0.25% w / v initiator standard solution; take 0.05 g of gelatin and put it into the initiator standard solution, heat in a 50℃ water bath in the dark until the gelatin is completely dissolved, stirring and shaking during the process to obtain a 0.5% w / v gelatin solution; take 0.2 g of AlgMA (50 kDa 1.2-1.4% w / v, 300 kDa 0.6-0.8% w / v) and put it into the 0.5% w / v gelatin solution, heat in a 50℃ water bath in the dark until the AlgMA is completely dissolved, stirring and shaking during the process to obtain a 2% w / v AlgMA and 0.5% w / v gelatin mixed solution.

[0081] Step 2 of the above scheme specifically involves: injecting 0.5 ml of the mixed solution prepared in step 1 into a custom cylindrical mold with a diameter of 15 mm and a height of 2 ml; irradiating the hydrogel mixed solution under a handheld 405 nm ultraviolet light source for 60–600 s; and obtaining viscoelastic hydrogels with different degrees of cross-linking through covalent cross-linking.

[0082] A method for preparing high-viscosity viscoelastic hydrogels (Alg-Gel) via ionic crosslinking.

[0083] Step 3: Prepare a serum-free DMEM solution containing 2% w / v sodium alginate (100kDa) and 0.5% w / v gelatin.

[0084] Step 4: Prepare serum-free DMEM solutions containing 24mM, 36mM, 56mM, and 80mM calcium chloride solutions, respectively.

[0085] Step 5: Serum-free DMEM solutions of 24mM, 36mM, 56mM, and 80mM calcium chloride solutions, and solutions of 2% w / v sodium alginate and 0.5% w / v gelatin were mixed at a 1:4 volume ratio in a 5ml syringe using a Luer lock at room temperature and rapidly deposited into a custom mold to obtain viscoelastic hydrogels with different ionic crosslinking concentrations. The chemical structure of the sodium alginate ionic crosslinked hydrogel is shown below. Figure 1 As shown in (b).

[0086] Step 3 specifically involves: measuring 20 ml of serum-free DMEM solution, adding 0.1 g of gelatin, and heating in a 40-50°C water bath for 15 min while stirring at 500 r / min; taking 0.4 g of sodium alginate and adding it to the gelatin standard solution, while stirring at 500 r / min and heating in a 50°C water bath until the alginate is completely dissolved.

[0087] Step 4 specifically involves preparing five 10ml portions of serum-free DMEM solution, adding 13mg, 27mg, 40mg, 62mg, and 89mg of calcium chloride respectively, and stirring until the calcium chloride is completely dissolved to obtain serum-free DMEM solutions of calcium chloride at concentrations of 24mM, 36mM, 56mM, and 80mM.

[0088] Step 5 specifically involves: taking 0.1 ml of 24 mM, 36 mM, 56 mM, and 80 mM calcium chloride serum-free DMEM solutions and 0.4 ml of a solution containing 2% w / v sodium alginate and 0.5% w / v gelatin, respectively, mixing them in a 5 ml syringe at room temperature using a Luer lock, and rapidly depositing them into a custom cylindrical mold with a diameter of 15 mm and a height of 2 mm. Viscoelastic hydrogels with different ion crosslinking concentrations are obtained through ion crosslinking.

[0089] An experimental method for obtaining viscoelastic hydrogels with the same initial modulus but different viscosities through two different crosslinking mechanisms.

[0090] Step 6: Fourier transform infrared spectroscopy was used to chemically verify and confirm the chemical structures and cross-linking mechanisms of the two proposed hydrogels, as follows: Figure 2 In the case of pure alginic acid, at 3269 cm⁻¹ -1 A broad absorption peak appears at 2930 cm⁻¹, corresponding to the presence of hydrogen-bonded OH groups, while a peak appears at 2930 cm⁻¹. -1 The peak is due to the CH stretching vibration, with the main absorption peak appearing at 1594 cm⁻¹. -1 and 1406cm -1 This is due to the asymmetric and symmetric stretching vibrations of COO-. These two peaks (1594 cm⁻¹) are... -1 and 1406cm -1 The peak α is the most useful characteristic peak for studying ionic crosslinking processes. Clearly, sodium alginate and Ca... 2+ During crosslinking, the asymmetric and symmetric -COO- vibrational peaks shift to lower wavenumbers, from 1594 cm⁻¹ to lower wavenumbers. -1 and 1406cm -1 Moved to 1598cm -1 and 1408cm -1 This may be due to Ca 2+ A strong electrostatic interaction exists between sodium alginate and the carboxyl group. After methacrylation, sodium alginate exhibits a high electrostatic activity at 1680 cm⁻¹.-1 The nearby peak is due to the C=C stretching vibration. Photocrosslinking of methacryloyl alginate leads to the opening of the C=C double bond in the methacryloyl group, hence the peak at 1680 cm⁻¹. -1 The nearby absorption peaks disappear, and photocrosslinking may cause COO2. - The chemical environment changed, leading to changes at 1594 cm⁻¹ -1 The nearby stretching vibration peak shifted to 1604 cm⁻¹ -1 Therefore, Fourier transform infrared spectroscopy was used to chemically verify and confirm the results. Figure 1 The paper proposes two types of hydrogels and their chemical structures and cross-linking mechanisms.

[0091] Step 7: Verify through rheological experiments that hydrogels with different moduli can be obtained by changing the UV irradiation time (covalent cross-linked hydrogel) and the ionic cross-linking concentration (ionic cross-linked hydrogel).

[0092] Rheological testing: Rheological tests were performed using an Anton Paar MCr 302 rheometer at 25°C using a 50mm parallel plate system with a fixed gap distance of 50µm. Hydrogels prepared with different UV irradiation times (60s, 120s, 300s, 600s) were loaded onto the fixation device, and time-scanned at a frequency of 1Hz with a strain of 1% were evaluated to assess the effect of 405nm UV irradiation time on the storage modulus (G') of the AlgMA-Gel hydrogel. Figure 3 As shown, G' increases with the increase of UV irradiation time, proving that the mechanical properties of AlgMA-Gel hydrogel can be adjusted by the irradiation time.

[0093] Rheological testing: Rheological tests were performed using an Anton Paar MCr 302 rheometer at 25°C using a 50 mm parallel plate system with a fixed gap distance of 50 μm. Hydrogels prepared with different calcium ion concentrations (24 mM, 36 mM, 56 mM, 80 mM) were loaded onto the fixation device, and time-scanning was performed at a frequency of 1 Hz with a strain of 1% to evaluate the effect of ion concentration on the storage modulus (G') of the ion-crosslinked hydrogel Alg-Gel. Figure 4 As shown, the storage modulus G' increases with increasing ion concentration, proving that the mechanical properties of Alg-Gel hydrogel can be adjusted by ion concentration.

[0094] Step 8: The initial elastic modulus of hydrogels with different light exposure times (covalent crosslinking) and ionic crosslinking concentrations were measured using a single-column mechanical testing machine compression test. In the elastic modulus test, the gel was compressed to 20% strain at a deformation rate of 1 mm / min, and the slope of the linear stress-strain curve between 10% and 15% strain was obtained. Each parameter was repeated 3 times.

[0095] Elastic modulus test: According to step 8, the elastic modulus of covalently cross-linked hydrogels and ionically cross-linked hydrogels with different ion concentrations under different light exposure times were tested, as shown in Table 1. It can be observed that the initial elastic modulus of covalently cross-linked hydrogel AlgMA-Gel increases with increasing light exposure time, and the initial modulus of ionically cross-linked hydrogel Alg-Gel increases with increasing ion concentration.

[0096] Table 1 Elastic modulus of covalently cross-linked hydrogels and ionically cross-linked hydrogels with different ion concentrations under different light exposure times.

[0097]

[0098] Step 9: Based on the experimental results of Step 8, three groups of hydrogels with comparable initial elastic moduli (covalently cross-linked hydrogels and ionicly cross-linked hydrogels with comparable initial elastic moduli) were selected. Their initial elastic moduli were: low (covalently cross-linked hydrogel with 2% AlgMA (300kDa 0.6%, 50kDa 1.4%) – 0.5% gel after 60s of light exposure and ionicly cross-linked hydrogel with 24mM ion concentration of Alg(100kDa) 2% – 0.5%), medium (covalently cross-linked hydrogel with 2% AlgMA (300kDa 0.6%, 50kDa 1.4%) – 0.5% gel after 600s of light exposure and ionicly cross-linked hydrogel with 36mM ion concentration of Alg(100kDa) 2% – 0.5%), and high (covalently cross-linked hydrogel with 2% AlgMA (300kDa 0.8%, 50kDa) after 300s of light exposure). 1.2% — Gel 0.5% and ion-crosslinked hydrogels with an ion concentration of 56 mM (Alg(100 kDa) 2% — Gel 0.5%). The initial moduli of the three groups of hydrogels with comparable initial moduli for both crosslinking mechanisms are as follows: Figure 5 As shown.

[0099] Step 10: Further, stress relaxation was tested for covalently cross-linked and ionicly cross-linked hydrogels with initial elastic moduli equivalent. In the stress relaxation measurement, the gel was compressed to 15% strain at a deformation rate of 1 mm / min, thus keeping the strain constant, and then the stress change over time was recorded. Figure 6As shown in (a), the initial elastic moduli of covalently cross-linked hydrogels and ionicly cross-linked hydrogels exhibit different stress relaxation characteristics. Covalently cross-linked hydrogels show more elastic stress relaxation, while ionicly cross-linked hydrogels show faster stress relaxation. Both ionicly cross-linked and photocross-linked hydrogels are viscoelastic materials, but photocross-linked hydrogels show less stress relaxation and are more elastic, as confirmed by the frequency independence of the shear storage modulus. Figure 6 (b) The ion (calcium ion) crosslinked hydrogel exhibits strong stress relaxation because the hydrogel reorganizes over time, a fact confirmed by the frequency dependence of the shear storage modulus. Figure 6 In (b), the reason for this is that the ionic crosslinking agent is reversible; when stress is applied to the hydrogel, the ionic crosslinking agent can dissociate and recombine.

[0100] Step 11: Viscosity testing of covalently cross-linked hydrogels and ionicly cross-linked hydrogels with comparable initial elastic moduli. The viscosities of the two hydrogels with comparable initial moduli after curing were tested at a fixed frequency (f = 1 Hz) and strain (γ = 1%). Figure 7 As shown.

[0101] Experimental methods for regulating neural stem cell behavior by viscoelastic hydrogel viscosity

[0102] Step 12: Verification of the effect of viscoelastic hydrogel viscosity on neural stem cell adhesion.

[0103] Step 12-1: Apply a quantitative amount (0.2 ml / well) of three groups of hydrogels with similar initial moduli and different viscosities (low, medium and high elastic moduli respectively) to a 24-well plate, cure them according to the methods in Step 2 and Step 5, and let them stand for 2 hours.

[0104] Step 12-2: Wash the hydrogel prepared in step 10-1 twice with serum-free culture medium containing 1% penicillin / streptomycin and equilibrate overnight.

[0105] Step 12-3: The cultured neural stem cells NE-4C are then subjected to a 10... 4 pcs / cm 2 Cells were seeded at a density of [insert density here] into 24-well plates and cultured for 24 hours. Then, Action-Tracker Green was diluted 1:40 with PBS containing 1% BSA and 0.1% Trion X-100 and stained for 45 minutes. Cells were then stained with DAPI working solution (10 μg / ml) for 10 minutes. During this period, cells were washed multiple times with PBS containing 0.1% Trion X-100 before and after staining. The stained cells were then imaged under a 40x objective lens using an inverted fluorescence microscope, and the cell adhesion area was analyzed using Image software. Figure 8In Figure (a), neural stem cells (NE-4C) adhere to two viscoelastic hydrogel substrates with initial elastic moduli of approximately 1.7 kPa, 4 kPa, and 7.2 kPa, respectively. It is evident that the viscosity of the viscoelastic matrix significantly affects the adhesion and spreading of neural stem cells. On substrates with higher viscosity, the spreading area of ​​neural stem cells increases, and more synaptic-like structures are formed. Cell adhesion area statistics are shown below. Figure 8 As shown in (b), under the same initial modulus, increasing the viscosity of the viscoelastic matrix can increase the cell adhesion area. Ionic cross-linked matrices exhibit faster stress relaxation and higher dynamic viscosity, while covalent cross-linked matrices show more stable but relatively lower viscosity. For high-viscosity ionic cross-linked matrices, the dynamic properties of ionic bonds enable cells to generate greater adhesion forces in a higher viscosity environment. Specifically, higher viscosity can prolong the contact time between the cell membrane and the matrix and increase the lifetime of integrin-ligand binding on the cell membrane at the molecular level. This prolonged contact time and increased molecular stability enhance cytoskeleton remodeling, thereby promoting the adhesion, spreading, and synapse formation of neural stem cells. For low-viscosity covalent cross-linked matrices, molecular bonds are subject to greater thermal perturbation, leading to reduced cell adhesion stability.

[0106] Step 13: Experimental verification of the effect of viscoelastic hydrogel viscosity on neural stem cell survival

[0107] Step 13-1: Apply a quantitative amount (0.2 ml / well) of three groups of hydrogels with similar initial moduli and different viscosities (low, medium and high elastic moduli respectively) to a 24-well plate, cure them according to the methods in Step 2 and Step 5, and let them stand for 2 hours.

[0108] Step 13-2: Wash the hydrogel prepared in step 10-1 twice with serum-free culture medium containing 1% penicillin / streptomycin and equilibrate overnight.

[0109] Step 13-3: NE-4C neural stem cells (2 × 10^4 cells per square centimeter) were seeded in 24-well plates with different solidified hydrogels and cultured at 37°C and 5% CO2 for 48 hours. Dead and live cells were stained with PI and Calcein AM, respectively. The stained cells were then imaged under a 10x objective lens using an inverted fluorescence microscope. After imaging, the number of live and dead cells was statistically analyzed using ImageJ software. Figure 9(a) shows fluorescence microscopy images of NE-4C neural stem cells on low-viscosity and high-viscosity viscoelastic matrices with an initial modulus of 4 kPa. The results indicate that the number of viable cells was significantly higher on the high-viscosity viscoelastic matrix than on the low-viscosity matrix. Furthermore, quantitative analysis of cell viability on low-viscosity and high-viscosity matrices with initial moduli of 1.8 kPa, 4 kPa, and 7.2 kPa is shown below. Figure 9 As shown in (b). The results showed that, under the same initial modulus conditions, the cell survival rate on the high-viscosity matrix was significantly higher than that on the low-viscosity matrix, increasing by 17.9%, 15.2%, and 19.5%, respectively. This phenomenon indicates that the viscosity of the matrix has an important influence on the survival of NE-4C neural stem cells, and that within a certain range, increasing the matrix viscosity helps to improve the cell survival rate.

[0110] This invention relates to viscoelastic hydrogels with the same initial modulus but different viscosities prepared through two crosslinking mechanisms, and an experimental verification method for the effect of viscosity on the behavior of neural stem cells. These methods can be used in tissue engineering, tissue repair, and pharmaceuticals.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a viscoelastic hydrogel for evaluating the effect of hydrogel viscosity on cell behavior, characterized in that, include: Covalently cross-linked sodium alginate-based viscoelastic hydrogels and ionically cross-linked sodium alginate-based viscoelastic hydrogels were prepared respectively. Among them, multiple sets of covalently cross-linked viscoelastic hydrogels were obtained according to different polymerization times; Based on different ion crosslinking concentrations, multiple sets of ion-crosslinked viscoelastic hydrogels were obtained; The initial elastic modulus of multiple covalently cross-linked viscoelastic hydrogels and ionicly cross-linked viscoelastic hydrogels were measured. Covalently cross-linked viscoelastic hydrogels and ionicly cross-linked viscoelastic hydrogels with similar initial elastic modulus were grouped together, and the viscosity of this group of hydrogels was tested. If the two viscoelastic hydrogels had different viscoelastic moduli, viscoelastic hydrogels with the same initial modulus but different viscoelastic ... The method for preparing the covalently cross-linked viscoelastic hydrogel includes: Methacrylamide alginate, gelatin, and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate were mixed evenly in a solvent to obtain a mixed solution; The mixed solution was polymerized under ultraviolet light to obtain a covalently cross-linked viscoelastic hydrogel; The preparation method of the ion-crosslinked viscoelastic hydrogel includes: Prepare a serum-free DMEM solution containing sodium alginate and gelatin; Prepare a serum-free DMEM solution containing calcium chloride; A serum-free DMEM solution containing calcium chloride and a serum-free DMEM solution containing sodium alginate and gelatin were mixed, and the resulting sediment was collected to obtain an ion-crosslinked viscoelastic hydrogel.

2. The method for preparing a viscoelastic hydrogel for evaluating the effect of hydrogel viscosity on cell behavior as described in claim 1, characterized in that, The mass ratio of the methacryloylated alginate, gelatin, and phenyl (2,4,6-trimethylbenzoyl)lithium phosphate is 2:0.5:0.25-0.

3.

3. The method for preparing a viscoelastic hydrogel for evaluating the effect of hydrogel viscosity on cell behavior as described in claim 1, characterized in that, The mass ratio of sodium alginate to gelatin is 2:0.

5.

4. The method for preparing a viscoelastic hydrogel for evaluating the effect of hydrogel viscosity on cell behavior as described in claim 1, characterized in that, The concentration of calcium chloride is 24~80 mM.

5. The application of the method described in any one of claims 1-4 in preparing viscoelastic hydrogels for evaluating the effect of hydrogel viscosity on cell behavior.

6. The application as described in claim 5, characterized in that, The cells in question are neural stem cells.

7. The application as described in claim 5, characterized in that, The cell behaviors referred to are cell adhesion and cell survival.

8. A method for evaluating the effect of viscoelastic hydrogel viscosity on neural stem cell adhesion and survival, comprising: The viscoelastic hydrogel prepared by the method of any one of claims 1-4 is solidified on a perforated plate, left to stand, washed, and a perforated plate with different solidified hydrogels is obtained. Neural stem cells were seeded onto well plates with different solidified hydrogels, cultured, and the cell adhesion area and the number of live and dead cells were statistically analyzed. The effect of viscoelastic hydrogel viscosity on the adhesion and survival of neural stem cells was evaluated based on the statistical results.

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