GM / PEG hydrogel scaffold as well as preparation method and application thereof

Abstract

The invention discloses a GM / PEG hydrogel scaffold and a preparation method and application thereof.The preparation method comprises the steps that gelatin is dissolved in a phosphate buffer solution, methacrylic anhydride is dropwise added for a reaction, a gelatin-methacrylic anhydride reaction solution is obtained, the phosphate buffer solution is added for standing overnight, supernate is taken for dialysis, and the GM / PEG hydrogel scaffold is obtained. The preparation method comprises the following steps: preparing a reaction solution, freeze-drying the reaction solution to obtain methacrylated gelatin, dissolving the methacrylated gelatin in PBS to obtain a methacrylated gelatin solution, mixing the methacrylated gelatin solution with a polyethylene glycol diacrylate solution, adding a photoinitiator to obtain a precursor solution, coating the surface of a balloon with the precursor solution to form a layer of precursor solution film, and curing under ultraviolet irradiation to form the GM / PEG hydrogel stent with a three-dimensional network structure. According to the preparation method disclosed by the invention, flexible regulation and control of mechanical properties are realized through the accurate proportion of the methacrylated gelatin and the polyethylene glycol diacrylate and a photo-crosslinking process, the process is stable, and the preparation method is suitable for large-scale production, has high strength, biological activity and controllable degradability, and has a wide prospect when being applied to an intravascular stent.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and in particular relates to a GM / PEG hydrogel scaffold and a preparation method and application thereof. Background Art

[0002] A vascular stent is a medical device used to treat vascular stenosis or blockage. It is typically implanted through minimally invasive surgery to prop open the narrowed or blocked vessel and restore blood flow, achieving the therapeutic purpose. Existing vascular stents include bare metal stents (BMS), drug-eluting stents (DES), biodegradable stents (such as polylactic acid and magnesium alloy stents), and covered stents.

[0003] However, traditional BMS stents have a high rate of restenosis and may trigger long-term inflammatory responses in the vessel wall. DES stents, on the other hand, pose risks of late thrombosis, long-term inflammatory responses, and issues with their drug delivery systems. Polylactic acid biodegradable stents also have mechanical deficiencies, such as thicker stent walls, weaker support, potential inflammation from degradation products, a degradation rate that is inconsistent with vascular repair, limited clinical application, and a higher risk of postoperative complications. Summary of the Invention

[0004] In order to overcome the above-mentioned deficiencies in the prior art, the main purpose of the present invention is to provide a method for preparing a GM / PEG hydrogel scaffold, which is formed by cross-linking methacrylated gelatin and polyethylene glycol diacrylate.

[0005] Another object of the present invention is to provide a GM / PEG hydrogel scaffold obtained by the aforementioned preparation method, which has the performance advantages of high strength, biological activity and controllable degradability.

[0006] Another object of the present invention is to provide an application of the GM / PEG hydrogel scaffold for preparing medical materials such as vascular stents.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing a GM / PEG hydrogel scaffold, comprising the following steps:

[0009] S1. Dissolving gelatin in phosphate buffer, then adding methacrylic anhydride dropwise. After the reaction is complete, a gelatin-methacrylic anhydride reaction solution is obtained;

[0010] S2, adding phosphate buffer to the gelatin-methacrylic anhydride reaction solution, letting it stand overnight, taking the supernatant for dialysis, and freeze-drying the resulting reaction solution to obtain methacrylated gelatin powder;

[0011] S3, dissolving methacrylated gelatin powder in PBS to obtain a methacrylated gelatin solution; mixing the polyethylene glycol diacrylate solution and the methacrylated gelatin solution, and adding a photoinitiator to the mixed solution to obtain a precursor solution;

[0012] S4. The precursor solution is coated on the surface of the balloon to form a precursor solution film, which is then cured under ultraviolet light to form a GM / PEG hydrogel scaffold with a three-dimensional network structure.

[0013] Furthermore, in step S2, the temperature of the overnight standing is 4°C, and the molecular weight cut-off range of the dialysis is 8000-14000Da.

[0014] Furthermore, in step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 3:1-7.

[0015] Furthermore, in step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 3:2; or 1:1; or 3:7.

[0016] Furthermore, in step S3, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

[0017] Furthermore, in step S4, the ultraviolet light irradiation conditions include: a wavelength of 365-405 nm, an irradiation intensity of 5-10 mW / cm 2 , the irradiation time is 60s.

[0018] The second aspect of the present invention further provides a GM / PEG hydrogel scaffold prepared by the preparation method of the GM / PEG hydrogel scaffold.

[0019] The third aspect of the present invention further provides the use of the GM / PEG hydrogel scaffold in the preparation of a vascular stent.

[0020] Compared with the prior art, the present invention provides a GM / PEG hydrogel scaffold and its preparation method and application. The GM / PEG hydrogel scaffold has high strength, bioactivity and controllable degradability. Through the precise ratio of methacrylated gelatin and polyethylene glycol diacrylate and the photocrosslinking process, the mechanical properties can be flexibly regulated to meet the mechanical requirements of different blood vessels. At the same time, methacrylated gelatin retains natural cell adhesion sites, combined with the hydrophilicity of polyethylene glycol diacrylate, while promoting the adhesion and proliferation of endothelial cells, it inhibits the proliferation of smooth muscle cells. During the preparation process, light-proof reaction, pH control and dialysis purification are carried out to ensure a high grafting rate, low impurity residues, stable process and suitability for large-scale production. The GM / PEG hydrogel scaffold is used for vascular stents and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of infrared spectrum characterization of GM / PEG hydrogel scaffold in the embodiment.

[0022] Figure 2 This is an electron microscopy characterization of the GM / PEG hydrogel scaffold in the example.

[0023] Figure 3 These are side views and cross-sectional views of the GM / PEG hydrogel scaffold before and after use in the examples.

[0024] Figure 4 Schematic diagram of the preparation process of GM / PEG hydrogel scaffold in the example.

[0025] Figure 5 This is the strain distribution diagram of the GM / PEG hydrogel scaffold at different times in the embodiment.

[0026] Figure 6 This is a distribution diagram of the compressive stress changes of the GM / PEG hydrogel scaffold in the embodiment.

[0027] Figure 7 Schematic diagram of the monitoring results of the fluid pressure change of the GM / PEG hydrogel scaffold in the embodiment.

[0028] Figure 8 Schematic diagram of the outer diameter measurement results of the GM / PEG hydrogel scaffold in the embodiment.

[0029] Figure 9 Schematic diagram of the test results of the liquid absorption capacity, degradation behavior and compressive mechanical properties of the GM / PEG hydrogel scaffold in the examples.

[0030] Figure 10 Schematic diagram of the stress change test and dynamic friction coefficient test results of the GM / PEG hydrogel scaffold in the embodiment.

[0031] Figure 11 Schematic diagram of the tensile and compressive mechanical properties test results of the GM / PEG hydrogel scaffold in the example.

[0032] Figure 12 Schematic diagram of the rheology and burst pressure test results of the GM / PEG hydrogel scaffold in the examples.

[0033] Figure 13 Schematic diagram of the structural changes of the GM / PEG hydrogel scaffold under different pressures in the embodiment.

[0034] Figure 14 Schematic diagram of the in vivo delivery demonstration and fluid dynamics simulation of the GM / PEG hydrogel scaffold in the examples.

[0035] Figure 15 This is a schematic diagram of the initial state of delivering the GM / PEG hydrogel scaffold into the blood vessel in the embodiment.

[0036] Figure 16 This is a schematic diagram of the balloon expansion state after the GM / PEG hydrogel scaffold is delivered into the blood vessel in the embodiment.

[0037] Figure 17 Schematic diagram of the completed state of delivering the GM / PEG hydrogel scaffold into the blood vessel in the embodiment; 1. Blood vessel; 2. Balloon catheter; 3. Hydrogel scaffold; 4. Balloon. DETAILED DESCRIPTION

[0038] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the following is a further detailed and complete description of the technical effects produced by the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, and not all of them. It should be pointed out that for those skilled in the art, other embodiments obtained without departing from the concept of the present invention are all within the scope of protection of the present invention.

[0039] Example 1

[0040] See also Figure 1-3 , proposed a GM / PEG hydrogel scaffold formed by cross-linking methacryloylated gelatin and polyethylene glycol diacrylate.

[0041] Methacrylated gelatin (GM) is a photocurable hydrogel produced by methacrylating the primary amine residues of lysine on gelatin molecules with methacrylic anhydride. Its molecular structure contains the RGD cell adhesion sequence, which provides cell adhesion sites. GM exhibits photocurability, excellent biocompatibility, biodegradability, and tunable mechanical properties.

[0042] Polyethylene glycol diacrylate (PEGDA) is a double-bond modified polyethylene glycol with acrylate groups at both ends of its molecular structure. It can form a three-dimensional network structure through photocrosslinking. It has photocurability, controllable mechanical properties and good biocompatibility, and is often used to prepare hydrogels.

[0043] The GM / PEG hydrogel scaffold, formed by cross-linking methacryloylated gelatin and polyethylene glycol diacrylate, has a three-dimensional reticular structure. This 3D reticular structure provides abundant cell adhesion sites, facilitating cell attachment and proliferation. This 3D reticular structure also imparts excellent mechanical properties, enabling it to better mimic the physiological environment of vascular tissue. By adjusting the ratio of GM to PEGDA, the elastic modulus and strength of the hydrogel can be manipulated, bringing it closer to the mechanical properties of native vascular tissue. The GM / PEG hydrogel scaffold exhibits excellent biocompatibility and does not induce a significant immune response.

[0044] See also Figure 1 , the characterization results of two protein / peptide materials (marked as SF and SSF in the figure) were obtained by infrared spectroscopy (FTIR). The horizontal axis is the wave number (cm -1 , characterizing the fingerprint of the absorption spectrum), the ordinate is the change in transmittance, corresponding to the common functional groups and absorption bands of proteins, we can see:

[0045] Amide I, II, and III bands: These bands (1700-1600 cm -1 , 1550cm -1 Around 1300-1200cm -1 (left and right) are the characteristic absorption peaks of the amide bond (-CONH-) on the main chain of proteins or polypeptides; it can be observed from the spectrum that SF (blue) and SSF (red) have obvious absorption in these areas, but with slight differences in peak intensity and peak position.

[0046] Sulfonic acid group (-SO3H) region: There is a clear sulfonic acid absorption band marked SSF in the figure, appearing at 1200-1000cm -1 This is because the introduction of sulfonic acid groups (green and pink parts in the figure) into the SSF molecular structure causes new characteristic peaks to appear in a specific wavenumber range or the peak intensity is significantly enhanced.

[0047] SF is the basic silk protein material, and typical protein amide absorption is mainly observed; SSF introduces new functional groups (-SO3H) on silk protein through chemical modification (such as sulfonation, etc.), so compared with SF, there will be absorption peaks related to sulfonic acid groups, and some absorption bands of the protein main chain will also be affected by chemical modification and change in intensity or peak position.

[0048] Through infrared spectral comparison: SSF (red spectrum) successfully introduced sulfonic acid groups on the basis of the original silk protein (SF, blue spectrum) and retained the amide structure of the protein main chain. The change, disappearance or regeneration of the infrared absorption peak helped to confirm the chemical structure modification of the material and the existence of functional groups, thereby explaining the success of the modification and the impact of the modification on the molecular structure.

[0049] See also Figure 2 This figure shows the surface morphology of three different materials: G / GM (gelatin / methacrylated gelatin), G / GM / P (gelatin / methacrylated gelatin / polyethylene glycol diacrylate), and G / GM / PS (gelatin / methacrylated gelatin / polyethylene glycol diacrylate-salting out), as well as the cross-sectional morphology and element distribution of G / GM / PS.

[0050] The surface of G / GM presents a larger pore structure; the pore structure of G / GM / P becomes denser, indicating that the surface morphology of the material changes after the addition of polyethylene glycol diacrylate; the pores of G / GM / PS are further refined, and the surface is more uniform, further improving the structure of the material.

[0051] The cross-sectional morphology of G / GM / PS shows that G / GM / PS has a uniform thickness, indicating that the preparation process is stable; the edge of the cross section is relatively flat, indicating that the structure of G / GM / PS is relatively dense and has good mechanical properties.

[0052] The four figures in the lower right corner show the distribution of the four elements (C, O, N, and Na) in the G / GM / PS cross-section. The scale is 200 μm. C (red) is evenly distributed and serves as the main component of the matrix; O (purple) is evenly distributed; N (green) is concentrated on the surface; and Na (cyan) is relatively sparsely distributed.

[0053] See also Figure 3 , showing the side view and cross-sectional view of the G / GM / PS hydrogel scaffold before and after use.

[0054] Example 2

[0055] See also Figure 4 , a preparation method of GM / PEG hydrogel scaffold is proposed, comprising the following steps:

[0056] S1. Dissolving gelatin in phosphate buffer, then adding methacrylic anhydride dropwise. After the reaction is complete, a gelatin-methacrylic anhydride reaction solution is obtained;

[0057] The specific implementation method is as follows: first, 10g of gelatin is dissolved in 100mL of phosphate buffered saline (PBS) at 55°C and stirred continuously until a uniform clear solution is formed to obtain a gelatin-methacrylic anhydride reaction solution. To ensure that methacrylic anhydride (MA) can fully react with the free amino groups in the gelatin macromolecules, 8mL of MA is added dropwise under light-proof and slightly alkaline conditions (pH 7.5-8.5). The addition process is carried out for 2.5-3.5 hours under sufficient stirring (rotation speed of 500-600rpm) to promote the directional grafting of MA on the gelatin molecular skeleton and to avoid the side reaction of incomplete cross-linking as much as possible.

[0058] S2. Add phosphate buffer to the gelatin-methacrylic anhydride reaction solution, let it stand overnight, take the supernatant and perform dialysis. The resulting reaction solution is freeze-dried to obtain methacrylated gelatin powder for later use;

[0059] The specific implementation method is as follows: when the reaction is completed, about 400 mL of PBS is quickly added to the gelatin-methacrylic anhydride reaction solution to dilute and terminate the reaction. At this time, the mixed solution is allowed to stand overnight at 4°C to promote sufficient sedimentation of the macromolecular polymer and relative separation of impurities. The next day, the supernatant is taken and dialyzed in deionized water using a dialysis bag with a molecular weight cut-off range of 8000-14000Da to remove unreacted MA, small molecule by-products and other low molecular weight impurities. To ensure the dialysis effect, the dialysate is replaced regularly until the conductivity and pH reach a stable state. Finally, the obtained gel solution is freeze-dried to obtain GM powder for standby use.

[0060] S3, mixing the polyethylene glycol diacrylate solution and the methacrylated gelatin solution to obtain a mixed solution, and adding a photoinitiator to the mixed solution to obtain a precursor solution;

[0061] The specific implementation method is as follows: First, a 20wt% PEGDA solution and a 50wt% GM solution are prepared separately in PBS, ensuring that each solution is fully swollen and clear. Subsequently, based on the expected mechanical and biological performance requirements, the GM and PEGDA solutions are accurately and quantitatively mixed in a volume ratio of 6:4 to obtain a mixed solution GM / PEG-1. To achieve the photocrosslinking reaction, 0.2wt% of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) is added to the mixture and evenly dispersed by stirring to obtain a precursor solution.

[0062] S4. The precursor solution is coated on the surface of the balloon to form a thin film of the precursor solution, which is then cured under ultraviolet light to form a GM / PEG hydrogel scaffold with a three-dimensional network structure.

[0063] The specific implementation method is as follows: for the hydrogel scaffold forming process, the precursor solution is evenly coated in situ on the surface of the pre-expanded and shape-stable balloon, and the balloon surface position is gently rolled or adjusted to form a uniform bubble-free precursor solution film. At this time, to ensure the integrity of the hydrogel layer, the excess precursor solution at both ends of the balloon is promptly removed to prevent excessive aggregation at the edge. Subsequently, under ultraviolet light irradiation (wavelength 365-405nm, illumination of about 5-10mW / cm 2 ) for 60 s to cause free radical photocrosslinking of the precursor solution, thereby rapidly solidifying and forming a GM / PEG hydrogel scaffold with a three-dimensional network structure.

[0064] Example 3

[0065] This example provides a method for preparing a GM / PEG hydrogel scaffold based on Example 2. The GM and PEGDA solutions in step S3 of Example 2 are accurately and quantitatively mixed in a volume ratio of 3:2, thereby adjusting the GM and PEGDA solutions to be accurately and quantitatively mixed in a volume ratio of 1:1 to obtain a mixed solution GM / PEG-2. The remaining steps are the same as those in Example 2.

[0066] Example 4

[0067] This example provides a method for preparing a GM / PEG hydrogel scaffold based on Example 2, wherein the GM and PEGDA solutions in step S3 of Example 2 are accurately and quantitatively mixed in a volume ratio of 3:2, and the GM and PEGDA solutions are accurately and quantitatively mixed in a volume ratio of 3:7, thereby obtaining a mixed solution GM / PEG-3. The remaining steps are the same as those in Example 2.

[0068] Example 5

[0069] See also Figure 5-13 This embodiment provides a GM / PEG hydrogel scaffold performance test based on Example 1.

[0070] 1. Monitoring of stress changes and fluid pressure changes during deformation: Please refer to Figure 5-7 , monitor the changes in the mechanical properties of hydrogel materials and internal or external fluid pressure, study the coupling relationship between hydrogels and fluid pressure during deformation, and evaluate their mechanical response in a fluid pressure environment.

[0071] First, monitor the changes in fluid pressure inside the hydrogel: calibrate the mechanical testing machine and pressure monitoring system, install the tensile fixture, and check that the pressure sensor and monitoring system are working properly; install the hydrogel sample into the tensile fixture using the same method as the tensile mechanical properties test. If the pressure sensor is pre-buried inside the sample, connect the sensor lead to the pressure monitoring system.

[0072] Set the cyclic tensile parameters, including cyclic waveform, cyclic frequency, strain amplitude, and number of cycles, in the mechanical testing machine control software. The parameter setting method is the same as for the cyclic compression performance test, but the test mode is cyclic tensile. Set the data acquisition parameters in the pressure monitoring system software, start the mechanical testing machine and pressure monitoring system simultaneously, and begin the cyclic tensile test and fluid pressure monitoring. The mechanical testing machine records the force-displacement data during the tensile process, and the pressure monitoring system simultaneously records the pressure sensor signal. Repeat the test on at least three parallel samples.

[0073] See also Figure 7Liquid pressure change (purple curve): The liquid pressure fluctuates periodically between 80 and 160 mm Hg, and the fluctuation frequency is high, indicating that periodic pressure is applied in the experiment; outer diameter change (orange curve): The outer diameter changes periodically between 3.06 and 3.09 mm. The change in outer diameter is synchronized with the change in liquid pressure, indicating that the material can respond quickly to pressure changes;

[0074] See also Figure 6 , showing the performance of G / GM / PS in the original state (Origin) and compression state (Compression). The simulation results show that the stress distribution of G / GM / PS in the compression state is uneven, and the color changes from blue to red, indicating that the values ​​in some areas have increased significantly.

[0075] See also Figure 5 This figure shows the strain distribution of G / GM / PS at different time points (0s, 20s, 40s, 60s, and 80s), with color used to represent the magnitude of strain. At 0s, the structure appears primarily blue, indicating a relatively uniform strain distribution and low overall strain levels, indicating a low initial strain state. At 20s, the color begins to transition to green, and strain begins to increase in certain areas, possibly indicating the structure is beginning to experience external forces, indicating a gradual increase in strain. At 40s, the color transitions further to yellow and red, indicating a more uneven strain distribution and continued strain increase. At 60s, most areas of the structure turn yellow and red, indicating further expansion of the strain distribution and reaching a high level. At 80s, the color distribution becomes more uniform, approaching red, indicating a stable strain distribution and a high overall strain level, indicating that stress has reached its maximum. The uniformity of the strain distribution indicates that G / GM / PS maintains good stability under load.

[0076] 2. External diameter measurement and testing: Please refer to Figure 8 The external diameter measurement results show the changes in the external diameter (ED) of three gel scaffolds, G / GM, G / GM / P, and G / GM / PS, in dry (purple bars) and wet (orange bars) states. The inset diagram illustrates the swelling process of the gel scaffold from dry to hydrated state, visually demonstrating the increase in ED as the scaffold absorbs water. The results demonstrate that the swelling properties and dimensional stability of the gel scaffold under hydrated conditions can be further manipulated through formulation modification.

[0077] 3. Liquid absorption capacity test: please refer to Figure 9 The left panel shows fluid absorption tests for three gel scaffolds: G / GM, G / GM / P, and G / GM / PS. The ordinate represents the adsorbed mass per unit mass (mg / mg), and the abscissa represents the absorption time (s). The inset shows a comparison of the macroscopic appearance of the gel scaffolds before and after immersion in simulated body fluid.

[0078] 4. Degradation behavior test: Please refer to Figure 9 Middle panel shows the residual weight evolution of three gel scaffolds (G / GM, G / GM / P, and G / GM / PS) over time (days). The inset shows typical photographs of the scaffold's appearance during immersion, illustrating the scaffold's structural stability at different time points.

[0079] 5. Compression mechanical properties test: please refer to Figure 9 The right figure shows the comparison of the compression force-strain curves of the gel scaffolds at different storage times (30 days, 60 days, and 90 days). The inset is the corresponding compression force bar graph statistical results (mean ± standard deviation), and statistical symbols are used to indicate significant differences.

[0080] 6. Stress change test at different time points: please refer to Figure 10 The left figure shows the stress-strain curves of three gel scaffolds, G / GM, G / GM / P and G / GM / PS, under tensile mode, showing the stress comparison of the three materials at specific time points. The stress of G / GM / PS is significantly higher than that of the other two materials. It also shows the self-expansion properties of the material in PBS (phosphate buffered saline) and its application in vascular stents.

[0081] 7. Dynamic friction coefficient test: please refer to Figure 10 The figure on the right shows a time-varying curve comparing the tribological performance of gel scaffolds made of different materials and formulations over a 600-second test. The inset on the left shows a photograph of the surface contact angle measurement (G / GM / PS, with an angle of approximately 43.9°±4.7°), while the three-dimensional surface topography of the material on the right visualizes the surface roughness and microstructure of the gel scaffold. The results indicate that the hydrophilicity of the gel scaffold (measured by the contact angle) and the surface microstructure (reflected in the three-dimensional topography) jointly influence its tribological performance, as evidenced by the relatively stable friction coefficient variation over the test. The results indicate that the scaffold surface is very smooth and does not cause blood flow disturbances.

[0082] 8. Tensile mechanical properties test: please refer to Figure 11 , determine the mechanical properties of hydrogel materials under uniaxial tensile load and evaluate the tensile properties of the materials.

[0083] Start the universal mechanical testing machine and calibrate the force and displacement sensors according to the equipment manual to ensure accurate test data. Carefully install the hydrogel sample vertically into the tensile fixture. Ensure that the sample axis is aligned with the tensile direction and that the clamping force is moderate to prevent the sample from slipping and to avoid pre-damage to the sample.

[0084] The following parameters were set in the testing machine control software: uniaxial stretching; displacement control; stretching speed of 1-10 mm / min; data acquisition frequency ≥ 10 Hz; and a preload force of 0.01 N.

[0085] After confirming that the parameter settings are correct, start the testing machine and begin the tensile test. The machine will stretch the sample at the set speed until it breaks. The machine automatically records the force (F) and displacement (ΔL) during the stretching process and generates a force-displacement curve. Repeat the test on at least 3-5 parallel samples to ensure the reliability and representativeness of the test results.

[0086] Based on the recorded force-displacement data, the stress (σ) and strain (ε) of each data point are calculated. The stress calculation formula is as follows:

[0087]

[0088] Where A0 is the initial cross-sectional area of ​​the sample neck, A0 = w × t.

[0089] The strain calculation formula is as follows:

[0090]

[0091] See also Figure 11 The upper left corner shows the stress-strain curves of three gel scaffolds, G / GM, G / GM / P and G / GM / PS, under tensile mode. The inset shows the sample state diagram before and after the experiment.

[0092] 9. Compression mechanical properties test: please refer to Figure 11 The lower left corner figure shows the determination of the mechanical properties of the hydrogel material under uniaxial compression load and the evaluation of the compression performance of the material.

[0093] Start the universal mechanical testing machine and calibrate the force sensor and displacement sensor according to the machine's operating manual. Place the hydrogel sample in the center of the machine's lower platen. Ensure that the top and bottom surfaces of the sample are parallel to the platen and that the sample is positioned firmly. If using lubricant, apply a thin layer evenly to the top and bottom surfaces of the sample.

[0094] The following parameters were set in the testing machine control software: uniaxial compression; displacement control; compression speed range of 1-5 mm / min; data acquisition frequency ≥ 10 Hz; set a preload force of 0.01 N; and 50% or 70% compressive strain value as the termination condition.

[0095] After confirming that the parameter settings are correct, start the compression test. The machine will compress the sample at the set speed until the set end condition is reached. The machine automatically records the force (F) and displacement (Δh) during the compression process and generates a force-displacement curve. Repeat the test on at least 3-5 parallel samples.

[0096] According to the recorded force-displacement data, the stress (σ) and strain (ε) of each data point are calculated and the stress-strain curve is drawn. The stress calculation formula is as follows:

[0097]

[0098] Where A0 is the initial cross-sectional area of ​​the sample,

[0099] The strain calculation formula is as follows:

[0100]

[0101] Where h0 is the initial height of the sample.

[0102] See also Figure 11 The lower left figure shows the compression force-strain curves of three gel scaffolds, G / GM, G / GM / P, and G / GM / PS, under compression mode. The inset shows the columnar comparative analysis of the compression force or compression modulus.

[0103] 10. Cyclic Compression Performance Test: Evaluate the mechanical properties of hydrogel materials under repeated cyclic compression loads and study their fatigue characteristics, recovery and energy dissipation characteristics.

[0104] Start the universal mechanical testing machine, calibrate the force sensor and displacement sensor according to the equipment operating manual, and install the compression fixture; place the hydrogel sample in the center of the lower pressure plate of the testing machine, using the same method as the compression mechanical properties test.

[0105] The following parameters were set in the testing machine control software: cyclic compression; strain control; cyclic waveform was either a sine wave or a triangle wave; cyclic frequency was 0.1-1 Hz; strain amplitude was 0%, 20%, and 30% compressive strain; and the number of cycles was 1, 25, 50, 75, and 100.

[0106] After confirming that the parameter settings are correct, start the testing machine to begin the cyclic compression test. The testing machine will perform cyclic loading according to the set parameters; the testing machine automatically records the force (F) and displacement (Δh) data during each cycle and generates a force-displacement curve for each cycle; repeat the test on at least 3 parallel samples.

[0107] The stress-strain curve for each cycle was plotted to observe the changes in the curve shape. The energy dissipation (Wd) for each cycle was calculated by integrating the area enclosed by the stress-strain curve for each cycle. Energy dissipation reflects the energy lost by the hydrogel during the cyclic compression process, which is converted into heat or other forms of energy.

[0108] Calculate the compression modulus for each cycle or at a certain interval (every 100 cycles) and analyze the trend of the compression modulus with the number of cycles to evaluate the fatigue properties and structural stability of the hydrogel. The compression modulus can be calculated by taking the slope of the stress-strain curve at the initial stage of each loading cycle (strain 0-10%).

[0109] After the cyclic compression, the residual deformation (plastic deformation) of the hydrogel sample was measured to evaluate the recovery of the hydrogel; the average value and standard deviation of the energy dissipation, compression modulus and residual deformation were calculated.

[0110] See also Figure 11 The figure on the lower right shows a comparison of the force-strain curves of a typical G / GM / PS gel scaffold after 1, 25, 50, 75, and 100 compression-relaxation cycles, as well as the macroscopic appearance of the scaffold in its initial form, after compression, and after relaxation. Figure 11 The radar chart in the upper right corner compares the comprehensive mechanical properties of the three gels in five aspects: strength, maximum strain, toughness, modulus and bearing capacity.

[0111] 11. Rheological testing: Study the viscoelastic behavior of hydrogel materials, measure their storage modulus (G') and loss modulus (G"), and evaluate the viscoelastic properties of the materials.

[0112] Start the rheometer and calibrate it according to the equipment operating manual, including the calibration of the torque sensor, displacement sensor and temperature control system. Select a suitable test geometry (parallel plate or cone plate) and install it on the rheometer, carefully placing the hydrogel sample in the center of the lower geometry of the rheometer. Adjust the upper geometry, slowly lower it to the sample surface, and set the appropriate test gap (Gap). The size of the gap is determined according to the sample thickness. For example, it is set to 90%-95% of the sample thickness to ensure that the sample is in full contact with the upper and lower geometries while avoiding excessive extrusion. For parallel plate geometry, it is recommended that the gap be slightly smaller than the sample thickness (the sample thickness is 1mm and the gap is set to 0.95mm).

[0113] Set the following parameters in the rheometer control software:

[0114] Oscillating frequency sweep: sweep frequency range 0.01-10 Hz or wider; frequency sweep at a strain amplitude of 1% or less; test temperature is room temperature (25°C) or body temperature (37°C); oscillating strain sweep; strain control; sweep strain range 0.01%-100% or wider; test frequency 1 Hz;

[0115] Steady-state shear flow: shear rate control; sweep shear rate range 0.01-100s -1 Or wider range; the test temperature is room temperature (25℃) or human body temperature (37℃).

[0116] After confirming that the parameter settings are correct, start the rheometer to begin the test. The rheometer will measure according to the set parameters; the rheometer automatically records the data during the test. The frequency sweep test records the storage modulus (G'), loss modulus (G"), complex viscosity (η*) and loss tangent (tanδ) at different frequencies; the strain sweep test records G' and G" at different strain amplitudes; the steady-state shear flow test records the viscosity at different shear rates. Repeat the test on at least 3 parallel samples.

[0117] Frequency sweep data analysis: Plot the storage modulus (G'), loss modulus (G"), and loss tangent (tanδ) as a function of frequency. Analyze the viscoelastic properties of the hydrogel. For example, when G'>G", the material primarily exhibits elastic solid properties; when G">G', the material primarily exhibits viscous liquid properties. tanδ = G" / G' reflects the relative ratio of the material's viscous and elastic components.

[0118] Strain sweep data analysis: Plot the storage modulus (G') and loss modulus (G") as a function of strain amplitude. Determine the hydrogel's linear viscoelastic range, that is, the strain range over which G' and G" remain constant. This linear viscoelastic range is crucial for selecting parameters for subsequent oscillation tests.

[0119] Steady-State Shear Flow Data Analysis: Plot viscosity (η) as a function of shear rate. Analyze the shear-thinning or shear-thickening behavior of the hydrogel. Most hydrogels exhibit shear-thinning behavior, meaning that viscosity decreases with increasing shear rate.

[0120] Results statistics and presentation: For frequency sweeps, G', G", and tanδ values ​​at a specific frequency (1 Hz) can be extracted for statistical analysis, and the mean and standard deviation can be calculated. For steady-state shear flow, viscosity values ​​at a specific shear rate can be extracted for statistical analysis.

[0121] See also Figure 12 The left figure shows the changes in the storage modulus (G') and loss modulus (G") of the three gel scaffolds in different frequency ranges; the lower right corner is a schematic diagram of the gel network structure, showing the cross-linking and hydration of the modified gel scaffold.

[0122] 12. Burst pressure test: Determine the maximum fluid pressure (burst pressure) that the hydrogel material can withstand and evaluate the pressure resistance of the material.

[0123] Assemble the burst pressure test device, ensuring that all components are tightly connected, the pipelines are unobstructed, and the pressure sensor is correctly installed. Check the sealing of the device to prevent pressure leakage. Fix the hydrogel sample to the sample fixture. The fixing method should be based on the sample shape and test device design. Seal the two ends of the sample to the pressure pipeline. Use sealing materials (O-rings or sealing tape) to enhance the sealing and ensure that the pressure is only applied to the effective area of ​​the sample.

[0124] Inject the pressure medium (deionized water) into the pressure line of the test device and remove the air in the line to ensure that the pressure medium fills the entire system. Set the parameters of the pressure control system (pressure increase rate range is 0.1-1MPa / s), start the pressure source to apply pressure to the hydrogel sample at the set pressure increase rate, and monitor the pressure value on the pressure display in real time.

[0125] Continue applying pressure until the hydrogel sample ruptures (develops cracks, holes, or leakage). Record the pressure at the moment of rupture, which is the burst pressure. The pressure sensor will accurately record the peak pressure at the moment of burst. Repeat the test on at least three parallel samples.

[0126] The bursting pressure value of each sample was recorded, and the average value and standard deviation of the bursting pressure were calculated to evaluate the discreteness and reliability of the hydrogel's pressure resistance.

[0127] See also Figure 12 In the figure on the right, three types of gel scaffolds are subjected to liquid-filled impact, and their burst pressures are measured and compared. The illustration is a schematic diagram of the test device and the operating process.

[0128] 13. Structural changes of G / GM / PS under different pressures: Please refer to Figure 13 , showing the structural changes of G / GM / PS under two different pressures (80 mmHg and 120 mmHg), where color is used to represent the magnitude of force (in Newton, N), and the color bar shows the range of force (from 0 to 2.5 N).

[0129] The G / GM / PS hydrogel scaffold exhibited certain deformation under a pressure of 80 mmHg, and the color distribution showed that the force was concentrated in certain areas, indicating that the material was subjected to greater force in these areas; under a pressure of 120 mmHg, the deformation of the G / GM / PS material was more significant, and the color distribution showed that the concentrated areas of force were more obvious, indicating that the material was subjected to greater force in these areas.

[0130] The G / GM / PS material exhibits certain deformation under both pressures, which indicates that it has self-expansion properties and can automatically adjust its shape under pressure.

[0131] Example 6

[0132] See also Figure 14-17 This example performs in vitro delivery demonstration and fluid dynamics simulation based on Example 1.

[0133] 1. In vitro delivery demonstration:

[0134] See also Figure 14-17 , showing the process from the start of delivering the GM / PEG hydrogel scaffold into the blood vessel to completion. Figure 15 Before the operation begins, the hydrogel scaffold 3 exists in its initial slender form. It is evenly and carefully coated on the surface of the balloon 4. Subsequently, the balloon 4 coated with the hydrogel scaffold 3 is placed on the balloon catheter 2. When preparing for vascular intervention, the hydrogel scaffold 3 is appropriately pressed against the surface of the balloon 4 to form a stable structure so that it will not shift or deform during the subsequent vascular passage. Then, the balloon catheter 2 with the hydrogel scaffold 3 is carefully delivered into the target blood vessel 1 by pushing the guide wire or catheter.

[0135] See also Figure 16 After reaching the predetermined position within the vessel 1, balloon 4 expands, pushing the hydrogel scaffold 3 from its initial elongated form to a fully expanded state. This transformation is not simply a physical expansion, but rather a rearrangement and adjustment of the hydrogel's internal structure, allowing it to better adapt to the structure of the vessel 1 after expansion.

[0136] Then, when balloon 4 begins to deflate, hydrogel scaffold 3, leveraging its unique material properties and pre-designed elastic memory, automatically rebounds under its own elasticity, adhering tightly and evenly to the inner wall of blood vessel 1. This adhesion not only provides mechanical support but also helps promote the growth and repair of endothelial cells, laying the foundation for long-term blood vessel health.

[0137] See also Figure 17 After confirming that the hydrogel scaffold 3 has been stably adhered to the inner wall of the blood vessel 1, the balloon catheter 2 is carefully removed to complete the entire surgical procedure, thereby bringing the best treatment effect and minimal trauma to the patient.

[0138] See also Figure 14 In the first two rows of images, we can see the morphology of the stent and its delivery process in the vascular model, as well as its expansion and fixation in the simulated or in vitro model. Figure 14 In the schematic diagram and actual photo on the upper left, you can see the initial shape of the stent. The stent is moderately pressed against the balloon surface so that it can stably pass through the blood vessel model during subsequent operations.

[0139] Figure 14As can be seen in the figure, the simulated blood vessel (rabbit blood vessel in vitro model) is fixed on the experimental platform, and then the balloon catheter with the hydrogel scaffold is delivered into the target blood vessel segment by pushing the guide wire or catheter.

[0140] After the outer sheath is removed, the hydrogel scaffold will automatically rebound and adhere to the inner wall of the blood vessel under the action of its own elasticity. Figure 14 In the film, we can see that in the simulated blood vessel, the hydrogel stent gradually changes from its initial slender form to a fully expanded form. The actual photo is marked with state changes such as "Insert->Self-expanding->Inside:vascular stents".

[0141] The expanded hydrogel scaffold adheres to the inner wall of the blood vessel. The interface morphology between the hydrogel scaffold and the blood vessel can be seen through photos or microscopic observation. Figure 14 Cross-sectional images of blood vessels and stents corresponding to dyeing or fluorescent labeling can be seen under a microscope, thereby evaluating the combination of the stent and the inner wall, cell attachment or tissue morphological changes.

[0142] Figure 14 In the center, a full cardiovascular simulation model (transparent chest cavity and major blood vessels) is used to conduct in vitro perfusion experiments with installed stents, simulating actual blood flow. "Before" and "After" comparison images illustrate the internal pathways within the vessel, the stent's position, and how blood or biomimetic fluid flows through the area before and after stent installation.

[0143] 2. Fluid mechanics simulation:

[0144] exist Figure 14 The color models and streamline diagrams on the right or below show the changes in blood flow distribution, velocity field, and wall shear force before and after stent installation.

[0145] First, the internal shape of the blood vessels in animals (or simulations) is acquired through CT, MRI, or even 3D scanning. Alternatively, a simplified computational model (e.g., a large arched artery with bifurcations) can be used to create a model in CAD software. The figure shows a 3D reconstruction of the aortic arch and several branching vessels, with the locations for stent placement noted.

[0146] In professional CFD software (ANSYS Fluent / CFX, COMSOL, OpenFOAM, etc.), the vascular lumen or the area containing the stent is meshed. To accurately capture the boundary layer flow around the stent, a finer mesh is added to the inner surface of the stent. Blood is often simplified as an incompressible, non-Newtonian fluid, with a flow rate or pressure waveform set at the inlet (proximal end) (or time-varying if pulsating); a fixed pressure or outflow distribution is often set at the outlet. No-slip boundary conditions are often set for the vessel wall and stent surface.

[0147] Figure 14 In the diagram, you can see two modes: "Control" and "Vascular stents": the flow comparison between the patient without stents and the patient with stents. Figure 14 You can see the streamlines diagram (velocity field distribution) and the thermal diagram of the wall shear stress (WSS) distribution;

[0148] from Figure 14 The middle color scheme shows that the highest blood flow velocity is located in the center of the vessel, decreasing near the inner wall. After stent placement, localized increases in velocity gradients or changes in flow distribution can also occur. Wall shear stress: WSS increases closer to vessel bends or the edge of the stent. Stent placement can sometimes cause localized increases or decreases in WSS, thereby affecting endothelial cell growth and re-endothelialization.

[0149] In the bottom two sets of diagrams, cross-sectional locations A, B, and C (or different vessel segments) are labeled. These cross-sectional locations can be used to compare velocity distribution, pressure distribution, or wall shear stress at different cross-sectional locations. By comparing the data from each cross-sectional area before and after stent placement, the effect of the stent on blood flow uniformity, shear stress, and potential thrombosis / restenosis risk can be assessed.

[0150] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a GM / PEG hydrogel scaffold, characterized in that: The following steps are involved: S1. Dissolving gelatin in phosphate buffer, then adding methacrylic anhydride dropwise. After the reaction is complete, a gelatin-methacrylic anhydride reaction solution is obtained; S2, adding phosphate buffer to the gelatin-methacrylic anhydride reaction solution, letting it stand overnight, taking the supernatant for dialysis, and freeze-drying the resulting reaction solution to obtain methacrylated gelatin powder; S3, dissolving methacrylated gelatin powder in PBS to obtain a methacrylated gelatin solution; mixing the polyethylene glycol diacrylate solution and the methacrylated gelatin solution, and adding a photoinitiator to the mixed solution to obtain a precursor solution; S4. The precursor solution is coated on the surface of the balloon to form a precursor solution film, which is then cured under ultraviolet light to form a GM / PEG hydrogel scaffold with a three-dimensional network structure.

2. The method for preparing the GM / PEG hydrogel scaffold according to claim 1, wherein In step S2, the temperature for standing overnight is 4°C, and the molecular weight cut-off range of the dialysis is 8000-14000Da.

3. The preparation method of GM / PEG hydrogel scaffold according to claim 1, characterized in that: In step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 3:1-7.

4. The method for preparing the GM / PEG hydrogel scaffold according to claim 3, wherein: In step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 3:

2.

5. The method for preparing the GM / PEG hydrogel scaffold according to claim 3, wherein: In step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 1:

1.

6. The method for preparing the GM / PEG hydrogel scaffold according to claim 3, wherein: In step S3, the volume ratio of the polyethylene glycol diacrylate solution to the methacrylated gelatin solution is 3:

7.

7. The method for preparing the GM / PEG hydrogel scaffold according to claim 1, wherein: In step S3, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

8. The method for preparing the GM / PEG hydrogel scaffold according to claim 1, wherein: In step S4, the ultraviolet light irradiation conditions include: wavelength of 365-405nm, irradiation intensity of 5-10mW / cm 2 , the irradiation time is 60s.

9. A GM / PEG hydrogel scaffold, characterized in that: The GM / PEG hydrogel scaffold is prepared by the preparation method of any one of claims 1 to 8, and is formed by cross-linking methacrylated gelatin and polyethylene glycol diacrylate.

10. Use of the GM / PEG hydrogel scaffold according to claim 9 in preparing a vascular stent.

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