Preparation method of heart failure treatment hydrogel with tissue repair and electrical signal conduction

Abstract

The present invention discloses a method for preparing a hydrogel for treating heart failure with tissue repair and electrical signal conduction, which relates to the field of medical material technology. The preparation method comprises the following steps: S1, dissolving a natural polysaccharide molecule with an amino group in water to obtain solution A; S2, dissolving a small molecule cross-linking agent with an aldehyde group in water to obtain solution B; S3, sulfation-modifying the natural macromolecule, then oxidatively polymerizing the EDOT monomer and the sulfated natural macromolecule, and dissolving them in water to obtain solution C; S4, culturing stem cells, separating, purifying, and freeze-drying to obtain a stem cell secretion group freeze-dried powder, dissolving it in water to obtain solution D; S5, mixing solution A, solution B, solution C, and solution D to obtain a hydrogel for treating heart failure. The hydrogel of the present invention has good biocompatibility under physiological conditions, can effectively match the electrical conductivity of the natural myocardium, and promote angiogenesis in the damaged area, thereby achieving the purpose of repairing heart damage.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a method for preparing a hydrogel for treating heart failure with the functions of tissue repair and electrical signal conduction. Background Art

[0002] Ischemic heart disease (IHD) is a leading cause of death and disability worldwide. Myocardial infarction (MI), caused by severe coronary artery obstruction, is one of the most severe and dangerous conditions, damaging both the myocardium and vascular vessels. Interruption of blood supply leads to the death of cardiomyocytes, but cardiomyocytes in adult mammals are terminally differentiated and have difficulty re-entering the cell cycle, limiting their endogenous regenerative pathways. The damaged myocardium is replaced by scar tissue, which lacks elasticity and electrical conductivity, leading to impaired cardiac systolic and diastolic function and disrupted electrophysiological signaling. Electrical coupling between CMs is achieved through gap junctions, but the non-conductive nature of fibrotic scar tissue leads to uncoupling of some surviving CMs in the infarcted area, creating a heterogeneous environment prone to arrhythmia induction. Therefore, restoring electrical and electromechanical connectivity within the damaged myocardium is crucial for restoring cardiac function after MI. Furthermore, promoting blood microcirculation through repairing or regenerating coronary arteries is a key challenge in achieving cardiac tissue regeneration.

[0003] In the early stages of infarction, reperfusion strategies implemented through thrombolysis or cardiac bypass can limit the infarct size and significantly reduce short-term mortality. However, reperfusion injury and the long-term irreversible myocardial remodeling process limit the effectiveness of reperfusion therapy. Emerging therapies represented by stem cell secretomes provide a reference approach for therapeutic angiogenesis after MI, but another major problem in MI treatment is that the uninterrupted contraction and relaxation of the ventricular wall and the flushing of blood flow will lead to a low retention rate of therapeutic substances in the infarct site, which greatly reduces the expected therapeutic effect and makes it difficult to provide long-term myocardial protection. Injectable hydrogels for the treatment of heart failure are advanced methods based on the concepts of tissue engineering and regenerative medicine. However, most hydrogels lack conductive properties, making it difficult to establish stable electrical coupling. In addition, single hydrogels lack biological activity, making it difficult to improve the adverse microenvironment of the damaged myocardium and unfavorable for inducing myocardial tissue repair and regeneration. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for preparing a hydrogel for treating heart failure with tissue repair and electrical signal conduction. The hydrogel has good biocompatibility under physiological conditions, can effectively match the electrical conductivity of the natural myocardium, and promote angiogenesis in the damaged area, thereby achieving the purpose of repairing heart damage, effectively solving the problems in the existing technology such as lack of conductive properties and being unfavorable for tissue repair and regeneration.

[0005] The present invention solves the above-mentioned technical problem with the following technical solution: a method for preparing a hydrogel for treating heart failure with tissue repair and electrical signal conduction capabilities is provided, comprising the following steps:

[0006] S1. Dissolve natural polysaccharide molecules with amino groups in water to obtain solution A;

[0007] S2, dissolving a small molecule cross-linking agent with an aldehyde group in water to obtain solution B;

[0008] S3, sulfation-modifying the natural macromolecule, and then oxidatively polymerizing the EDOT monomer and the sulfated natural macromolecule in an aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C;

[0009] S4. Cultivating stem cells, separating, purifying, and freeze-drying to obtain stem cell secretory group freeze-dried powder, which is dissolved in water to obtain solution D;

[0010] S5. Mix solution A, solution B, solution C, and solution D to obtain a hydrogel for treating heart failure with the capabilities of tissue repair and electrical signal conduction.

[0011] Furthermore, in step S1, the natural polysaccharide molecule with amino groups is carboxymethyl chitosan.

[0012] Furthermore, the concentration of solution A is 3-7 wt %, specifically 3 wt %, 4 wt %, 5 wt %, 6 wt % and 7 wt %.

[0013] Furthermore, the concentration of solution A is 5 wt%.

[0014] Furthermore, in step S2, the small molecule cross-linking agent with an aldehyde group is four-arm polyethylene glycol succinimidyl glutarate.

[0015] Furthermore, the concentration of solution B is 5-8 wt %, specifically 5 wt %, 6 wt %, 7 wt % and 8 wt %.

[0016] Furthermore, the concentration of solution B is 7 wt %.

[0017] Furthermore, step S3 specifically includes the following steps:

[0018] S31. Dispersing the natural macromolecule in N,N-dimethylformamide, then adding aminosulfonic acid, reacting at 70-90°C for 2 h, and dialyzing against water to obtain the sulfated natural macromolecule;

[0019] S32. Disperse the sulfated natural macromolecule and EDOT monomer in water under nitrogen atmosphere and ice bath conditions, and mix well to obtain solution 1;

[0020] S33, dissolving ammonium persulfate and ferric sulfate in water to obtain solution 2;

[0021] S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

[0022] Furthermore, in step S31, the natural macromolecule is nanocellulose.

[0023] Furthermore, in step S31, the mass ratio of the natural macromolecule to aminosulfonic acid is 5-10:1; such as 10:1, 8:1 and 5:1.

[0024] Furthermore, in step S32, the mass ratio of the sulfated natural macromolecule to the EDOT monomer is 0.5-2:1; such as 0.5:1, 1:1 and 2:1.

[0025] Furthermore, in step S32, the concentration of sulfated natural macromolecules in solution 1 is 1 wt%.

[0026] Furthermore, in step S32 and step S33, the mass ratio of ammonium persulfate to ferric sulfate is 40:1, and the concentration of ammonium persulfate is 20 wt%.

[0027] Furthermore, in step S32 and step S33, the mass ratio of ammonium persulfate to sulfated nanofibers is 1:2.

[0028] Furthermore, in step S34, the volume ratio of solution 1 to solution 2 is 10:1.

[0029] Further, step S4 specifically includes the following steps:

[0030] S41. Culture and passage human umbilical cord-derived mesenchymal stem cells using complete mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity.

[0031] S42. Wash the 6th to 10th passage mesenchymal stem cells with PBS, then incubate them in a serum-free and phenol red-free stem cell-specific basal medium for 4 h, wash them once with serum-free medium, and then culture them for 48 h.

[0032] S43. After the incubation is completed, the culture medium supernatant is collected, and the dead cells are removed by the first round of centrifugation, and the cell debris are removed by the second round of centrifugation. The mixture is filtered and freeze-dried to obtain the stem cell secretion group freeze-dried powder, which is dissolved in water to obtain solution D.

[0033] Furthermore, in step S43, the first round of centrifugation is performed at 300-800 xg for 10-20 min, and the second round of centrifugation is performed at 1000-5000 xg for 10-20 min.

[0034] Furthermore, in step S43, the first round of centrifugation was performed at 500 x g for 15 min, and the second round of centrifugation was performed at 3000 x g for 15 min.

[0035] Further, the solution was filtered through a 0.22 μm filter membrane.

[0036] Furthermore, in step S5, the volume ratio of solution A, solution B, solution C and solution D is 8:2:1:1.

[0037] Furthermore, in step S5, the concentrations of solution A (carboxymethyl chitosan aqueous solution), solution B (four-arm polyethylene glycol succinimidyl glutarate solution), solution C (PEDOT:sulfated nanofibers), and solution D (stem cell secretome) are 3-7 wt%, 5-8 wt%, 0.01-0.05 wt%, and 0.2-0.5 wt%, respectively.

[0038] The present invention also provides a heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities, which is prepared by the preparation method of the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities.

[0039] The present invention also provides the use of the above-mentioned heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities in the preparation of a drug for treating heart failure after myocardial infarction.

[0040] The present invention has the following beneficial effects:

[0041] 1. The preparation conditions of the present invention are mild, the gelation is rapid, and the preparation method is simple. The resulting hydrogel has excellent injectability, electrical signal conduction performance, and angiogenesis-promoting ability. It can match the electrophysiological activity of natural myocardium, deliver stem cell secretion groups with angiogenesis-promoting activity, promote myocardial electrical coupling and blood supply reconstruction, and achieve the goal of myocardial tissue repair and regeneration.

[0042] 2. This invention utilizes a novel conductive polymer PEDOT doping method, distinct from traditional polystyrene sulfonic acid (PSS) doping. Sulfated natural macromolecules are used as dispersants and dopants for PEDOT, effectively avoiding the acidic degradation products introduced by PSS and further improving the biocompatibility of the conductive polymer. The highly conductive PEDOT:sulfated nanofibers and angiogenic stem cell secretomes are loaded into a Schiff base-crosslinked hydrogel system. The resulting composite hydrogel exhibits excellent injectability, electrical signal conduction properties, and angiogenic potential. It matches the electrophysiological activity of native myocardium, delivering the angiogenic stem cell secretomes, promoting myocardial electrical coupling and revascularization, and ultimately achieving the goal of myocardial tissue repair and regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The gelation time and injectability test diagrams of the hydrogel obtained in Example 1; a) is the gelation time measurement, b) is the injectability test;

[0044] Figure 2 The microscopic morphology of the hydrogels obtained in Comparative Example 1 and Example 1 under a scanning electron microscope;

[0045] Figure 3 Rheological behaviors of the hydrogels obtained in Comparative Example 1 and Example 1; a) shows the change in hydrogel viscosity with shear rate, and b) shows the change in storage modulus G' / loss modulus G'' with strain;

[0046] Figure 4 This is an atomic force microscope image of the PEDOT:sulfated nanofibers obtained in Example 1;

[0047] Figure 5 The conductivity values ​​of the hydrogels obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4;

[0048] Figure 6 The effects of the hydrogel extracts obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 on the cell viability of rat cardiomyocytes H9C2;

[0049] Figure 7 The effects of the hydrogel extracts obtained in Comparative Example 1, Example 1, Example 4, and Example 5 on the angiogenic behavior of human umbilical vein endothelial cells; a) is a bright-field micrograph of tubular structures formed by human umbilical vein endothelial cells on Matrigel; b) is a qualitative statistical analysis of the number of nodes in the capillary-like structures; c) is a qualitative statistical analysis of the total length of the capillary-like structures;

[0050] Figure 8 Graph showing the results of animal therapeutic ultrasound experiments on the hydrogels obtained in Comparative Example 1 and Example 1. DETAILED DESCRIPTION

[0051] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0052] Example 1

[0053] A hydrogel for treating heart failure with tissue repair and electrical signal conduction capabilities, the preparation method of which comprises the following steps:

[0054] S1. Dissolve carboxymethyl chitosan in water to obtain solution A (5 wt%);

[0055] S2, dissolving four-arm polyethylene glycol succinimidyl glutarate in water to obtain solution B (7 wt %);

[0056] S3. Sulfation modification of the natural macromolecule, followed by oxidative polymerization of the EDOT monomer and the sulfated natural macromolecule in aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C (0.02 wt%).

[0057] S4. Cultivating stem cells, separating, purifying, and freeze-drying to obtain stem cell secretory group freeze-dried powder, which was dissolved in water to obtain solution D (0.2 wt%);

[0058] S5. Solution A, solution B, solution C, and solution D were mixed in a mass ratio of 8:2:1:1 to obtain CPS@secretome, a hydrogel for treating heart failure with tissue repair and electrical signal conduction capabilities.

[0059] Wherein, step S3 specifically includes the following steps:

[0060] S31. Disperse 1.6 g of nanocellulose in 20 mL of N,N-dimethylformamide, then add 0.2 g of aminosulfonic acid, react at 80 °C for 2 h, and dialyze against water for 3 days to obtain sulfated nanocellulose.

[0061] S32. Under nitrogen atmosphere and ice bath conditions, disperse 0.5 g of sulfated nanocellulose and 600 μL of EDOT monomer in 50 mL of water and mix well to obtain solution 1.

[0062] S33. Dissolve 1.2 g of ammonium persulfate and 30 mg of ferric sulfate in 5 mL of water to obtain Solution 2.

[0063] S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water for 3 days, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

[0064] Step S4 specifically includes the following steps:

[0065] S41. Culture and passage human umbilical cord-derived mesenchymal stem cells using complete mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity.

[0066] S42. Wash the 6th to 10th passage mesenchymal stem cells with PBS, then incubate them in a serum-free and phenol red-free stem cell-specific basal medium for 4 h, wash them once with serum-free medium, and then culture them for 48 h.

[0067] S43. After incubation, collect the culture supernatant and centrifuge at 500 x g for 15 min to remove dead cells. Then centrifuge at 3000 x g for 15 min to remove cell debris. Filter through a 0.22 μm filter and freeze-dry to obtain stem cell secretome freeze-dried powder, which is then dissolved in water to obtain solution D.

[0068] Example 2

[0069] A hydrogel for treating heart failure with tissue repair and electrical signal conduction capabilities, the preparation method of which comprises the following steps:

[0070] S1. Dissolve carboxymethyl chitosan in water to obtain solution A (5 wt%);

[0071] S2, dissolving four-arm polyethylene glycol succinimidyl glutarate in water to obtain solution B (7 wt %);

[0072] S3. Sulfation modification of the natural macromolecule, followed by oxidative polymerization of the EDOT monomer and the sulfated natural macromolecule in aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C (0.02 wt%).

[0073] S4. Cultivating stem cells, separating, purifying, and freeze-drying to obtain stem cell secretory group freeze-dried powder, which was dissolved in water to obtain solution D (0.2 wt%);

[0074] S5. Solution A, solution B, solution C, and solution D are mixed in a mass ratio of 8:2:1:1 to obtain a hydrogel for treating heart failure with the capabilities of tissue repair and electrical signal conduction.

[0075] Wherein, step S3 specifically includes the following steps:

[0076] S31, dispersing 1.6 g of nanocellulose in 20 mL of N,N-dimethylformamide, then adding 0.16 g of aminosulfonic acid, reacting at 80 °C for 2 h, and dialyzing against water for 3 days to obtain sulfated nanocellulose;

[0077] S32. Under nitrogen atmosphere and ice bath conditions, disperse 0.5 g of sulfated nanocellulose and 750 μL of EDOT monomer in 50 mL of water and mix well to obtain solution 1.

[0078] S33. Dissolve 1.2 g of ammonium persulfate and 30 mg of ferric sulfate in 5 mL of water to obtain Solution 2.

[0079] S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water for 3 days, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

[0080] Step S4 specifically includes the following steps:

[0081] S41. Culture and passage human umbilical cord-derived mesenchymal stem cells using complete mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity.

[0082] S42. Wash the 6th to 10th passage mesenchymal stem cells with PBS, then incubate them in a serum-free and phenol red-free stem cell-specific basal medium for 4 h, wash them once with serum-free medium, and then culture them for 48 h.

[0083] S43. After incubation, collect the culture supernatant and centrifuge at 500 x g for 15 min to remove dead cells. Then centrifuge at 3000 x g for 15 min to remove cell debris. Filter through a 0.22 μm filter and freeze-dry to obtain stem cell secretome freeze-dried powder, which is then dissolved in water to obtain solution D.

[0084] Example 3

[0085] A hydrogel for treating heart failure with tissue repair and electrical signal conduction capabilities, the preparation method of which comprises the following steps:

[0086] S1. Dissolve carboxymethyl chitosan in water to obtain solution A (5 wt%);

[0087] S2, dissolving four-arm polyethylene glycol succinimidyl glutarate in water to obtain solution B (7 wt %);

[0088] S3. Sulfation modification of the natural macromolecule, followed by oxidative polymerization of the EDOT monomer and the sulfated natural macromolecule in aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C (0.02 wt%).

[0089] S4. Cultivating stem cells, separating, purifying, and freeze-drying to obtain stem cell secretory group freeze-dried powder, which was dissolved in water to obtain solution D (0.2 wt%);

[0090] S5. Solution A, solution B, solution C, and solution D are mixed in a mass ratio of 8:2:1:1 to obtain a hydrogel for treating heart failure with the capabilities of tissue repair and electrical signal conduction.

[0091] Wherein, step S3 specifically includes the following steps:

[0092] S31, disperse 1.6 g of nanocellulose in 20 mL of N,N-dimethylformamide, then add 0.32 g of aminosulfonic acid, react at 80 °C for 2 h, and dialyze against water for 3 days to obtain sulfated nanocellulose;

[0093] S32. Under nitrogen atmosphere and ice bath conditions, disperse 0.5 g of sulfated nanocellulose and 200 μL of EDOT monomer in 50 mL of water and mix well to obtain solution 1.

[0094] S33. Dissolve 1.2 g of ammonium persulfate and 30 mg of ferric sulfate in 5 mL of water to obtain Solution 2.

[0095] S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water for 3 days, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

[0096] Step S4 specifically includes the following steps:

[0097] S41. Culture and passage human umbilical cord-derived mesenchymal stem cells using complete mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity.

[0098] S42. Wash the 6th to 10th passage mesenchymal stem cells with PBS, then incubate them in a serum-free and phenol red-free stem cell-specific basal medium for 4 h, wash them once with serum-free medium, and then culture them for 48 h.

[0099] S43. After incubation, collect the culture supernatant and centrifuge at 500 x g for 15 min to remove dead cells. Then centrifuge at 3000 x g for 15 min to remove cell debris. Filter through a 0.22 μm filter and freeze-dry to obtain stem cell secretome freeze-dried powder, which is then dissolved in water to obtain solution D.

[0100] Example 4

[0101] The preparation method of the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities is the same as that of Example 1, except that the concentration of solution C in step S4 is 0.3 wt %.

[0102] Example 5

[0103] The preparation method of the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities is the same as that of Example 1, except that the concentration of solution C in step S4 is 0.5 wt %.

[0104] Comparative Example 1

[0105] A hydrogel, the preparation method of which comprises the following steps:

[0106] A 5 w / v% carboxymethyl chitosan (CMC) aqueous solution A and a 7 w / v% four-arm polyethylene glycol succinimidyl glutarate solution B were prepared, and solutions A and B were mixed in a volume ratio of 8:2 to obtain a hydrogel.

[0107] Comparative Example 2

[0108] A hydrogel, the preparation method of which comprises the following steps:

[0109] A 5 w / v% carboxymethyl chitosan (CMC) aqueous solution A and a 7 w / v% four-arm polyethylene glycol succinimidyl glutarate solution B were prepared. The purchased PEDOT:PSS aqueous dispersion was diluted to a 0.02 w / v% solution C. Solutions A, B, and C were mixed in a volume ratio of 8:2:1 to obtain a hydrogel.

[0110] Comparative Example 3

[0111] A hydrogel, the preparation method of which comprises the following steps:

[0112] S1. Dissolve carboxymethyl chitosan in water to obtain solution A (5 wt%);

[0113] S2, dissolving four-arm polyethylene glycol succinimidyl glutarate in water to obtain solution B (7 wt %);

[0114] S3. Sulfation modification of the natural macromolecule, followed by oxidative polymerization of the EDOT monomer and the sulfated natural macromolecule in aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C (0.02 wt%).

[0115] S5. Solution A, solution B, and solution C are mixed in a mass ratio of 8:2:1 to obtain a hydrogel.

[0116] Wherein, step S3 specifically includes the following steps:

[0117] S31. Disperse 1.6 g of nanocellulose in 20 mL of N,N-dimethylformamide, then add 0.2 g of aminosulfonic acid, react at 80 °C for 2 h, and dialyze against water for 3 days to obtain sulfated nanocellulose.

[0118] S32. Under nitrogen atmosphere and ice bath conditions, disperse 0.5 g of sulfated nanocellulose and 600 μL of EDOT monomer in 50 mL of water and mix well to obtain solution 1.

[0119] S33. Dissolve 1.2 g of ammonium persulfate and 30 mg of ferric sulfate in 5 mL of water to obtain Solution 2.

[0120] S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water for 3 days, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

[0121] Test example

[0122] 1. Gelation time and injectability

[0123] The gelation time of the hydrogel obtained in Example 1 was detected by the vial inversion method: the three precursor solutions obtained in Example 1 were mixed in a vial, the state of the mixture in the vial was observed, and the time required for the hydrogel to reach a stable state was recorded.

[0124] The injectability of the hydrogel prepared in Example 1 was tested by needle injection. The results are as follows: Figure 1 shown.

[0125] Depend on Figure 1 The results show that -NH2 on the carboxymethyl chitosan molecular chain and -CHO on the four-arm polyethylene glycol succinimidyl glutarate rapidly crosslink under mild conditions to form dynamic covalent imine bonds, a process known as a Schiff base reaction. The Schiff base-crosslinked hydrogel exhibits excellent shear thinning properties. When passed through a 27 G needle, the viscosity of the hydrogel decreases under shear stress, demonstrating excellent injectability. Furthermore, the hydrogel maintains a stable gel state after shear stress is unloaded.

[0126] 2. Hydrogel Micromorphology

[0127] The hydrogel micromorphology was observed by scanning electron microscopy: The hydrogels obtained in Example 1 and Comparative Example 1 were freeze-dried and then quenched with liquid nitrogen. The freeze-dried samples were sprayed with gold and their micromorphology was observed using a scanning electron microscope. The experimental results are shown in Figure 2. Figure 2 shown.

[0128] Depend on Figure 2 It can be seen that the hydrogel obtained in Example 1 has a porous three-dimensional network microstructure.

[0129] 3. Rheological behavior of hydrogel

[0130] The rheological properties of the hydrogel obtained in Example 1 of the present invention were tested by an MCR302 rheometer. The test was conducted at 37°C using a double concentric cylinder geometry with a gap of 1 mm. The flow characteristics of the hydrogel were investigated by a rotation experiment. The shear rate was measured from 0 s -1 to 100 s -1 The viscosity curve of the hydrogel under different conditions was measured, and then an oscillation experiment was performed to determine the deformation characteristics of the hydrogel. In the strain sweep test mode, the frequency was constant at 1 Hz, and the strain was set to increase from 0.01% to 100%. The relationship curve between the strain and the storage modulus G' and loss modulus G'' was measured. The experimental results are shown in Figure 2. Figure 3 shown.

[0131] Depend on Figure 3It can be seen that the hydrogel obtained in Example 1 has a three-dimensional network structure and has good shear thinning properties.

[0132] 4. Micromorphology of PEDOT: Sulfated Nanofibers

[0133] The microscopic morphology of the PEDOT-doped sulfated nanofibers obtained in Example 1 of the present invention was observed by atomic force microscopy. The experimental results are as follows: Figure 4 shown.

[0134] Depend on Figure 4 It can be seen that the microstructure of the PEDOT:sulfated nanofibers obtained in Example 1 is as follows: hydrophobic PEDOT agglomerates serve as the core, and the sulfated nanofibers with good hydrophilicity serve as the dispersant and dopant.

[0135] 5. Conductive properties of hydrogel

[0136] The resistivity of the hydrogels obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1 was measured by a four-probe method, and the conductivity of the hydrogels was calculated by the formula. The experimental results are shown in FIG. Figure 5 shown.

[0137] Depend on Figure 5 It can be seen that after the PEDOT:sulfated nanofibers of the present invention are loaded into the hydrogel, the conductivity of the blank hydrogel is greatly improved, and there is no significant difference in conductivity from the hydrogel with the same concentration of commercially available PEDOT:PSS added.

[0138] 6. Biocompatibility of hydrogels

[0139] The biocompatibility of the hydrogels was evaluated by measuring the effects of the hydrogels obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1 on cell viability. The experimental results are shown in FIG. Figure 6 shown.

[0140] Depend on Figure 6 It can be seen that after 48 h of extraction, the cell viability in the extract of Comparative Example 2 decreased significantly due to the presence of PEDOT:PSS, while there was no significant difference between Comparative Example 3 and Comparative Example 1. In Example 1, the presence of the stem cell secretion group promoted cell proliferation and significantly enhanced cell viability.

[0141] VII. In vitro angiogenesis performance of hydrogels

[0142] The in vitro angiogenesis performance of the hydrogel obtained in Example 1 was tested by an in vitro angiogenesis experiment: the hydrogels prepared in Example 1, Example 4, Example 5 and Comparative Example 1 were placed in 10 mL of serum-free culture medium and extracted for 48 h, and human umbilical vein endothelial cells were seeded in a well plate. The original culture medium was replaced with serum-free culture medium, the extract of Comparative Example 1, the culture medium supplemented with endothelial growth factor ECGF, and the hydrogel extracts of Example 1, Example 4 and Example 5, respectively. After culturing for 24 h, the cells were seeded on Matrigel to observe angiogenesis. The experimental results are shown in FIG. Figure 7 shown.

[0143] Depend on Figure 7 It can be seen that there was no obvious tubular network formation in the blank control group and the comparative example 1 group; in the experimental group, with the increase of secretome concentration, the density of the vascular network increased, and the number of nodes and total tube length of the Example 4 group increased significantly compared with the control group, and there was no significant difference with the ECGF positive control group.

[0144] 8. In vivo effectiveness testing of hydrogel

[0145] In order to study the effect of heart failure treatment hydrogel with tissue repair and electrical signal conduction on repairing heart damage in vivo, a rat myocardial infarction disease model was established by permanently ligating the left anterior descending branch of the heart; the hydrogels obtained in Example 1 and Comparative Example 1 were implanted into the rats with myocardial infarction model for treatment. 30 μL of hydrogel was injected into the infarct center and the infarct edge with a 27G needle. On the 28th day, the rats with successfully modeled myocardial infarction were subjected to echocardiography, and the heart sections were subjected to Masson staining. The M-mode echocardiography test results are as follows: Figure 8 As shown. Among them, Figure 8 In the sham operation group, the skin was cut open to expose the heart, but no ligation or injection was performed.

[0146] Depend on Figure 8 It can be seen that compared with the myocardial infarction model group, the ventricular wall systolic-diastolic motion of the Example 1 group was significantly improved, proving that its heart repair function was good.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel for treating heart failure with tissue repair and electrical signal conduction, characterized in that: The following steps are involved: S1. Dissolve natural polysaccharide molecules with amino groups in water to obtain solution A; S2, dissolving a small molecule cross-linking agent with an aldehyde group in water to obtain solution B; S3, sulfation-modifying the natural macromolecule, and then oxidatively polymerizing the EDOT monomer and the sulfated natural macromolecule in an aqueous solution to obtain PEDOT: The sulfated natural macromolecule is dissolved in water to obtain solution C; wherein the sulfate-modifying the natural macromolecule is aminosulfonic acid, and the natural macromolecule is nanocellulose; S4. Cultivating stem cells, separating, purifying, and freeze-drying to obtain stem cell secretory group freeze-dried powder, which is dissolved in water to obtain solution D; S5. Mix solution A, solution B, solution C, and solution D to obtain a hydrogel for treating heart failure with the capabilities of tissue repair and electrical signal conduction.

2. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 1, wherein: In step S1, the natural polysaccharide molecule with an amino group is carboxymethyl chitosan, and the concentration of solution A is 3-7 wt%.

3. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 1, wherein: In step S2, the small molecule cross-linking agent with an aldehyde group is four-arm polyethylene glycol succinimidyl glutarate, and the concentration of solution B is 5-8 wt%.

4. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 1, wherein: Step S3 specifically includes the following steps: S31. Dispersing the natural macromolecule in N,N-dimethylformamide, then adding aminosulfonic acid, reacting at 70-90°C for 2 h, and dialyzing against water to obtain the sulfated natural macromolecule; S32. Disperse the sulfated natural macromolecule and EDOT monomer in water under nitrogen atmosphere and ice bath conditions, and mix well to obtain solution 1; S33, dissolving ammonium persulfate and ferric sulfate in water to obtain solution 2; S34. Solution 2 was added to solution 1, and then reacted at 0°C for 48 h, dialyzed against deionized water, and freeze-dried to obtain PEDOT:sulfated nanofibers; which were dissolved in water to obtain solution C.

5. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 4, wherein: In step S31, the natural macromolecule is nanocellulose.

6. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 4, wherein: In step S31, the mass ratio of the natural macromolecule to aminosulfonic acid is 5-10:

1.

7. The method for preparing the heart failure treatment hydrogel with tissue repair and electrical signal conduction capabilities according to claim 1, wherein: Step S4 specifically includes the following steps: S41. Culture and passage human umbilical cord-derived mesenchymal stem cells using complete mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity. S42. Wash the 6th to 10th passage mesenchymal stem cells with PBS, then incubate them in a serum-free and phenol red-free stem cell-specific basal medium for 4 h, wash them once with serum-free medium, and then culture them for 48 h. S43. After the incubation is completed, the culture medium supernatant is collected, and the dead cells are removed by the first round of centrifugation, and the cell debris are removed by the second round of centrifugation. The mixture is filtered and freeze-dried to obtain the stem cell secretion group freeze-dried powder, which is dissolved in water to obtain solution D.

8. The method for preparing the hydrogel for treating heart failure with tissue repair and electrical signal conduction according to claim 1, wherein: In step S5, the volume ratio of solution A, solution B, solution C and solution D is 8:2:1:

1.

9. The hydrogel for treating heart failure with tissue repair and electrical signal conduction obtained by the method for preparing the hydrogel for treating heart failure with tissue repair and electrical signal conduction according to any one of claims 1 to 8.

10. Use of the heart failure therapeutic hydrogel with tissue repair and electrical signal conduction capabilities according to claim 9 in the preparation of a drug for treating post-myocardial infarction heart failure.

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