Conductive hydrogel loaded with adipose-derived mesenchymal stem cells as well as preparation method and application of conductive hydrogel

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to adipose-derived mesenchymal stem cell loaded conductive hydrogel and a preparation method and application thereof, the preparation method comprises the following steps: step S1, after hyaluronic acid is subjected to oxidation treatment, dopamine is grafted to oxidized hyaluronic acid, and oxidized hyaluronic acid-dopamine is obtained; step S2, carrying out amination treatment on gelatin to obtain aminated gelatin; s3, mixing the oxidized hyaluronic acid-dopamine with the aminated gelatin, and carrying out Schiff base crosslinking, so as to obtain hydrogel; and step S4, loading the adipose tissue-derived stromal cells by using the hydrogel. Dopamine is grafted on an oxidized hyaluronic acid macromolecular chain through amidation reaction, the hydrophilicity and the electrical property of the macromolecular chain are improved, a catechol structure is provided to provide possibility for iron ion coordination, the tissue adhesion, the injectability, the mechanical property and the electrical conductivity of the hydrogel are improved, and the hydrogel can be applied to the field of biomedical materials. After being applied to a spinal cord injury rat model, the functional recovery can be promoted.

Description

Technical Field

[0001] The present invention relates to a conductive hydrogel loaded with adipose mesenchymal stem cells, a preparation method thereof and an application thereof, belonging to the technical field of biomedical materials. Background Art

[0002] After spinal cord injury (SCI), due to the interruption of signal conduction within the brain-spinal cord circuit, motor dysfunction and sensory loss occur below the injury site. To solve the problem of interrupted nerve electrical signals, a comprehensive approach of regulating the microenvironment and restoring signal conduction is very promising for effective tissue repair after SCI. In recent years, conductive hydrogels have been developed and widely used in tissue repair, which are usually prepared by combining conductive substances and non-conductive hydrogels. Conductive hydrogels can regulate the microenvironment of the spinal cord by mimicking the high electrical conductivity of the spinal cord, reconstruct the signal conduction pathway of damaged nerves, and thus promote nerve regeneration.

[0003] Hyaluronic acid (HA) is a polysaccharide derived from natural extracellular matrix (ECM). Due to its inhibitory effect on glial scar formation, it has been proven beneficial for spinal cord tissue repair. HA hydrogels can produce materials with a highly porous structure and viscoelastic properties close to natural CNS tissues, but the lack of adhesiveness limits the bridging effect of HA.

[0004] In summary, improving the adhesiveness of conductive hydrogels, endowing them with good injectability and better conductivity, is of great significance and promise for the treatment of spinal cord injury. Summary of the Invention

[0005] In view of the above technical problems of the prior art, the present invention provides a conductive hydrogel loaded with adipose mesenchymal stem cells, a preparation method thereof and an application thereof.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: One of the objectives of the present invention is to provide a preparation method of a conductive hydrogel loaded with adipose mesenchymal stem cells, comprising the following steps: Step S1, after oxidizing hyaluronic acid, graft dopamine onto oxidized hyaluronic acid (OHA) to obtain oxidized hyaluronic acid-dopamine; Step S2, perform amination treatment on gelatin to obtain aminated gelatin; Step S3, mix the oxidized hyaluronic acid-dopamine obtained in step S1 with the aminated gelatin obtained in step S2, and perform Schiff base crosslinking to obtain a hydrogel; Step S4, load adipose mesenchymal stem cells into the hydrogel.

[0007] The hydrogel design of the present invention combines a double-network structure: dynamic coordination bonds (Fe 3+-catechol) and covalent crosslinking (Schiff base bond), thus enhancing the structural integrity and functional properties. First, Fe 3+ The dynamic reversibility of the Fe-dopamine coordination bond endows the hydrogel with self-healing ability, which is crucial for maintaining structural integrity after long-term implantation. Second, the introduction of dopamine not only enhances the tissue adhesion of the hydrogel, but also endows the material with conductivity due to the conductivity of dopamine and its binding to iron ions. This provides a biomimetic microenvironment for the conduction of nerve electrical signals. In addition, the double-network structure achieves a balance between mechanical properties and injectability through the synergistic effect of covalent crosslinking and ionic coordination, solving the problems of insufficient mechanical strength or unstable structure after injection of traditional hydrogels. Compared with the gelation formed by simple iron ion coordination, the double-network structure also significantly reduces the amount of iron ions required for gelation, thus improving the biosafety of the hydrogel.

[0008] In the context of spinal cord injury (SCI), oxidative stress plays an important role in the secondary injury process. Fe 3+ can serve as a catalytic center for redox buffering, facilitating the scavenging of excessive reactive oxygen species (ROS) and reducing oxidative damage to nerve tissue. This dual function, namely structural coordination and redox regulation, makes the Fe 3+ -based hydrogel of the present invention particularly promising for SCI repair, providing both mechanical support and a biochemical protection mechanism. How to effectively incorporate iron ions into the hydrogel while reducing its molar concentration to minimize potential iron-related toxicity remains a challenging issue. On this basis, how to design a hydrogel with injectable self-healing properties is also one of the effective means to enhance the clinical translational application of hydrogels. The conductive hydrogel of the present invention achieves injectable safety. This hydrogel uses Schiff base and ionic coordination bonds to coordinate iron ions. The double-network structure not only ensures mechanical strength but also enables efficient iron ion coordination, significantly reducing the amount of iron ions required and improving the safety of the hydrogel.

[0009] The nervous system is a complex tissue with high electrical activity. Electrical stimulation plays a crucial role in the growth and development process of nerve cells and the functional regulation mechanism. Research shows that appropriate electrical stimulation can regulate the physiological functions of nerve cells, promote the growth and extension of neurites, and enhance signal transmission between nerve cells. The conductive hydrogel of the present invention can transmit electrical signals to cells, simulate the electrophysiological environment of the nervous system, provide a microenvironment closer to the physiological state for the growth and differentiation of nerve cells, and further enhance the therapeutic effect of stem cells.

[0010] Based on the above technical solutions, the present invention can also be improved as follows: Further, in the step S1, the specific steps of the oxidation treatment of hyaluronic acid include: adding hyaluronic acid into deionized water, stirring until completely dissolved, adding sodium periodate and continuing to stir, and finally adding ethylene glycol to terminate the reaction. After dialysis, it is freeze-dried to obtain oxidized hyaluronic acid; the mass ratio of the hyaluronic acid, sodium periodate, ethylene glycol to deionized water is: 0.2 - 0.3:0.10 - 0.15:0.10 - 0.15:2 - 3.

[0011] Further, in the step S1, the specific steps of dopamine grafting include: dissolving oxidized hyaluronic acid in deionized water, adjusting the pH to 5 - 6, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and stirring for activation; adding dopamine hydrochloride, stirring and reacting for 24 h - 30 h, and after dialysis, it is freeze-dried to obtain oxidized hyaluronic acid-dopamine (OHA-DA); the mass ratio of the oxidized hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide to dopamine hydrochloride is: 8 - 10:6.6 - 7.0:1.95 - 2.00:4 - 5.

[0012] Further, in the step S1, the oxidation treatment of hyaluronic acid is carried out under dark conditions, and the reaction time is 4 h - 5 h; the dopamine grafting reaction is carried out under a N2 atmosphere.

[0013] Further, in the step S2, the specific steps of gelatin amination include: dissolving gelatin in PBS buffer, dispersing it evenly, adding ethylenediamine, adjusting the pH to 5 - 6, and then adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to start the reaction. After the reaction is completed, it is dialyzed and then freeze-dried for standby; the mass ratio of the gelatin, PBS buffer, ethylenediamine to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is: 0.5 - 1.0:10 - 20:0.8 - 1.0:0.23 - 0.30.

[0014] Further, the concentration of the PBS buffer is 0.1 M - 0.2 M. Gelatin needs to be heated and dispersed when dissolved in the PBS buffer. The temperature of the gelatin amination reaction is 35°C - 40°C, and the reaction time is 24 h - 30 h.

[0015] Further, in the step S3, ferric chloride is used as a cross-linking agent to coordinate with catechol to form a gel; the mixing volume ratio of oxidized hyaluronic acid-dopamine to aminated gelatin is 10:1 - 20:1, the concentration of ferric chloride is 2.5 mM - 3 mM, and the addition amount is 10% - 20% of the system.

[0016] In a common hydrogel gelation system, the addition amount of FeCl3 in 1 mL of the gelation system is about 50 μL (1.25 mg / mL), and the hydrogel system of the present invention can reduce the addition amount of FeCl3 in 1 mL of the gelation system by about 2 / 3.

[0017] Further, in the step S4, the loading of adipose mesenchymal stem cells is carried out after sterilization and removal of free metal ions.

[0018] Further, in the step S4, the specific steps of sterilization and removal of free metal ions include: after the gel is formed, it is washed with PBS buffer 3 to 5 times, 10 minutes each time, and ultraviolet irradiation is added during this period; soaked in absolute ethanol 3 to 5 times, 15 minutes each time, and ultraviolet irradiation is added during this period; soaked in 75% ethanol solution for half an hour, and ultraviolet irradiation is added during this period; washed with PBS buffer 3 to 5 times, 10 minutes each time, and ultraviolet irradiation is added during this period. The gel is changed to a new well, soaked in PBS / complete medium for 3 days, after aspirating the supernatant, it is washed with PBS / complete medium and then freeze-dried for standby. At this time, the free ions are basically removed completely.

[0019] Dopamine (DOPA) is a neurotransmitter, a chemical substance that can be used to help cells transmit impulses. It contains a large number of catechol groups and is soluble in aqueous solution. The catechol groups have adhesion properties to a variety of surfaces. The dopamine groups can undergo a complexation reaction with Fe 3+ under alkaline conditions, and the color of the solution changes from dark green to purple-red.

[0020] Gelatin is a natural polymer material, and its structure is similar to that of biological tissue structures. Therefore, it has good biocompatibility. As a natural water-soluble and biodegradable polymer material, its advantage is that the degradation products are easily absorbed without causing inflammatory reactions.

[0021] The preparation method of the present invention grafts dopamine onto the oxidized hyaluronic acid polymer chain by amidation reaction, improves the hydrophilicity and electrical properties of the polymer chain, and provides a catechol structure to provide the possibility for the coordination of iron ions. The present invention utilizes the Schiff base reaction between amino gelatin and oxidized hyaluronic acid and the coordination of iron ions with catechol to achieve double-network gelation. The conductive hydrogel prepared by this method can improve the mechanical properties and conductivity of the hydrogel. Compared with the gelation formed by simple iron ion coordination, the double-network gelation system of the present invention can reduce the addition amount of iron ions and the biological toxicity brought by them, and has good injectability and tissue adhesion.

[0022] The second object of the present invention is to provide a conductive hydrogel loaded with adipose mesenchymal stem cells prepared by the above preparation method.

[0023] The conductive hydrogel loaded with adipose mesenchymal stem cells of the present invention has both injectability, conductivity and neuroactivity, and has good porous structure, tissue adhesion, swelling property and mechanical properties similar to those of central nerve tissue. On this good basis, the hydrogel of the present invention is also loaded with adipose mesenchymal stem cells. Compared with other conductive gels, it can increase the regeneration of neurons at the injury site and promote the nerve repair at the injury site by synergistically conducting the conductive effect of the gel. The good injectability increases the potential for future clinical translation.

[0024] The third object of the present invention is to provide an application of the above-mentioned conductive hydrogel loaded with adipose mesenchymal stem cells, which is mainly used for preparing drugs or scaffolds for nerve tissue regeneration treatment.

[0025] Compared with the prior art, the beneficial effects of the present invention include: (1) The method of the present invention does not require expensive reagents and can be prepared only by conventional polymer materials; the preparation method is simple, the reaction conditions are mild, and the biocompatibility is good.

[0026] (2) The method of the present invention optimizes and improves the performance of the gel by using an ionic coordination bond and a Schiff base bond double-network gelation system, which is easy to control, and the double-network gelation system significantly reduces the amount of iron ions in the gel system.

[0027] (3) By adding dopamine, the hydrogel of the present invention has better adhesion, and the prepared gel has good injectability, conductivity and the ability to load adipose mesenchymal stem cells, and has good application prospects and research value for spinal cord injury and nerve tissue recovery. Description of the Drawings

[0028] Figure 1 It is a scanning electron microscope morphology diagram of the conductive hydrogel; Figure 2 It is a display of the conductivity and self-healing performance of the gel; Figure 3 It is a display of the injectability and tissue adhesion of the hydrogel; Figure 4 It is the CCK8 cell toxicity test result of the gel extract on PC12 cells; Figure 5 It is a fluorescence image of live and dead cell staining of the gel extract on ADSC cells.

[0029] Figure 6 It is the nerve function recovery of the injury site in the blank control group and the gel group after spinal cord injury in rats. Detailed Embodiments

[0030] The principles and features of the present invention are described below with reference to the drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1 A conductive hydrogel loaded with adipose mesenchymal stem cells, and the specific preparation steps are as follows: (1) After oxidizing hyaluronic acid, graft dopamine onto oxidized hyaluronic acid to obtain oxidized hyaluronic acid-dopamine, which specifically includes the following steps: ① Add 10 g of hyaluronic acid to 100 mL of deionized water, stir until completely dissolved, then add 5 g of sodium periodate to the solution, react for 4 h under dark conditions, add 5 mL of ethylene glycol to terminate the reaction, dialyze for three days, and then lyophilize to obtain oxidized hyaluronic acid for standby.

[0032] ② Dissolve 8 g of oxidized hyaluronic acid in deionized water, adjust the pH to 5, then add 6.6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.95 g of N-hydroxysuccinimide, stir evenly, activate for 30 min under N2 atmosphere; add 4 g of dopamine hydrochloride, stir and react for 24 h, then dialyze and lyophilize to obtain oxidized hyaluronic acid-dopamine.

[0033] (2) Amination treatment of gelatin, which specifically includes the following steps: Dissolve 5 g of gelatin in 100 mL of PBS buffer (0.1 M), heat and disperse evenly, add 8 mL of ethylenediamine, adjust the pH to 5 with hydrochloric acid, then add 2.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 35 °C for 24 h, dialyze and then lyophilize to obtain aminated gelatin for standby.

[0034] (3) Preparation of a conductive hydrogel scaffold, and prepare a hydrogel scaffold through Schiff base condensation reaction and ionic coordination, which specifically includes the following steps: Respectively take oxidized hyaluronic acid-dopamine and aminated gelatin, dissolve them in deionized water according to 20 wt%, then mix oxidized hyaluronic acid-dopamine and aminated gelatin at a ratio of 10:1, carry out Schiff base crosslinking, and use ferric chloride as a crosslinking agent to coordinate with catechol to form a gel, wherein the concentration of ferric chloride is 2.5 mM and the addition amount is 10% of the system.

[0035] The high-magnification scanning electron micrograph of the conductive hydrogel obtained in this example after lyophilization is as Figure 1 shown. From the Figure 1 presented structure, it can be seen that the hydrogel prepared in this example presents a porous structure after lyophilization, which is suitable for the loading of stem cells.

[0036] Example 2 A conductive hydrogel loaded with adipose mesenchymal stem cells, and the specific preparation steps are as follows: (1) After oxidizing hyaluronic acid, dopamine is grafted onto oxidized hyaluronic acid to obtain oxidized hyaluronic acid-dopamine, which specifically includes the following steps: ① Dissolve 15 g of hyaluronic acid in 100 mL of deionized water. After stirring until completely dissolved, add 5 g of sodium periodate to the solution. After reacting for 4 h under dark conditions, add 5 mL of ethylene glycol to terminate the reaction. After dialysis for three days, lyophilize to obtain oxidized hyaluronic acid for standby.

[0037] ② Dissolve 8 g of oxidized hyaluronic acid in deionized water. After adjusting the pH to 5, add 6.8 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 g of N-hydroxysuccinimide, stir evenly, and activate for 30 min under an N2 atmosphere; add 4.5 g of dopamine hydrochloride, stir and react for 24 h, then dialyze and lyophilize to obtain oxidized hyaluronic acid-dopamine.

[0038] (2) Amination treatment of gelatin, which specifically includes the following steps: Dissolve 5 g of gelatin in 100 mL of PBS buffer (0.1 M), heat and disperse evenly, add 9 mL of ethylenediamine, adjust the pH to 5 with hydrochloric acid, then add 2.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 35 °C for 24 h, dialyze and lyophilize to obtain aminated gelatin for standby.

[0039] (3) Preparation of a conductive hydrogel scaffold. A hydrogel scaffold is prepared through Schiff base condensation reaction and ion coordination, which specifically includes the following steps: Respectively take oxidized hyaluronic acid-dopamine and aminated gelatin, dissolve them in deionized water at 20 wt%, then mix oxidized hyaluronic acid-dopamine and aminated gelatin at a ratio of 15:1, conduct Schiff base crosslinking, and use ferric chloride as a crosslinking agent to coordinate with catechol to form a gel, where the concentration of ferric chloride is 2.8 mM and the addition amount is 15% of the system.

[0040] The conductivity and self-healing property of the conductive hydrogel obtained in this example are as Figure 2 shown, and it can be seen from Figure 2 that the hydrogel prepared in this example has good conductive ability and self-healing performance.

[0041] The present invention uses the syringe extrusion method to evaluate the injectability of the hydrogel. Load the hydrogel sample prepared in this example into a 5 mL syringe, and extrude the gel from a 22 G needle (inner diameter 0.41 mm) by manually applying pressure (the results are shown in Figure 3). During the experiment, it was observed that the gel showed good flow characteristics during extrusion, was able to form a continuous and uniform extrusion flow, and no obvious flow blockage or fracture occurred. It is worth noting that the extruded gel was able to maintain its original three-dimensional network structure and morphological integrity, and no obvious structural damage or dispersion occurred. This result indicates that the prepared hydrogel has good injectability, and its rheological properties can meet the application requirements of minimally invasive injection therapy. The injectability and tissue adhesiveness of the conductive hydrogel obtained in this example are as Figure 3 shown, and it can be seen from Figure 3 that the hydrogel prepared in this example has good injectability and tissue adhesiveness.

[0042] Comparative Example 1 A conductive hydrogel, the specific preparation steps are as follows: (1) Oxidize hyaluronic acid, which specifically includes the following steps: Add 10 g of hyaluronic acid and 100 mL of deionized water, stir until completely dissolved, then add 5 g of sodium periodate to the solution, react for 4 h under dark conditions, add 5 mL of ethylene glycol to terminate the reaction, dialyze for three days and then freeze-dry to obtain oxidized hyaluronic acid for standby.

[0043] (2) Amination treatment of gelatin, which specifically includes the following steps: Dissolve 5 g of gelatin in 100 mL of PBS buffer (0.1 M), heat and disperse evenly, add 8 mL of ethylenediamine, adjust the pH to 5 with hydrochloric acid, then add 2.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 35 °C for 24 h, dialyze and then freeze-dry to obtain aminated gelatin for standby.

[0044] (3) Preparation of hydrogel, prepare the hydrogel through Schiff base reaction, which specifically includes the following steps: Take oxidized hyaluronic acid and aminated gelatin, dissolve them in deionized water according to 20 wt%, then mix oxidized hyaluronic acid and aminated gelatin in a ratio of 3:2, and carry out Schiff base cross-linking to form a gel.

[0045] Compared with Example 2, this comparative example did not introduce dopamine groups, and this system does not have good conductivity.

[0046] Example 3 A conductive hydrogel loaded with adipose mesenchymal stem cells, the specific preparation steps are as follows: (1) After oxidizing hyaluronic acid, graft dopamine onto oxidized hyaluronic acid to obtain oxidized hyaluronic acid-dopamine, which specifically includes the following steps: ① Dissolve 10 g of hyaluronic acid in 100 mL of deionized water. After stirring until completely dissolved, add 7.5 g of sodium periodate to the solution. After reacting for 4 h under dark conditions, add 7.5 mL of ethylene glycol to terminate the reaction. After dialysis for three days, lyophilize to obtain oxidized hyaluronic acid for standby.

[0047] ② Dissolve 8 g of oxidized hyaluronic acid in deionized water. After adjusting the pH to 6, add 7 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 g of N-hydroxysuccinimide, and stir evenly. Activate for 30 min under an N2 atmosphere; add 5 g of dopamine hydrochloride, stir and react for 24 h, then dialyze and lyophilize to obtain oxidized hyaluronic acid-dopamine.

[0048] (2) Amination treatment of gelatin, specifically including the following steps: Dissolve 5 g of gelatin in 100 mL of PBS buffer (0.1 M), heat and disperse evenly, add 10 mL of ethylenediamine, adjust the pH to 5 with hydrochloric acid, then add 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 35 °C for 24 h, dialyze and lyophilize to obtain aminated gelatin for standby.

[0049] (3) Preparation of a conductive hydrogel scaffold. A hydrogel scaffold is prepared through Schiff base condensation reaction and ion coordination, specifically including the following steps: Respectively take oxidized hyaluronic acid-dopamine and aminated gelatin, dissolve them in deionized water at 20 wt%, then mix oxidized hyaluronic acid-dopamine and aminated gelatin in a ratio of 20:1, perform Schiff base crosslinking, and use ferric chloride as a crosslinking agent to coordinate with catechol to form a gel. Among them, the concentration of ferric chloride is 3 mM, and the addition amount is 20% of the system.

[0050] (4) Sterilization of the hydrogel and treatment of free metal ions: First, wash the hydrogel 3 times with PBS buffer, soak for 10 minutes each time and then aspirate and discard the clear water, and add ultraviolet irradiation during this period; soak in absolute ethanol 3 times, 15 minutes each time, and add ultraviolet irradiation during this period; soak in 75% ethanol for half an hour, and add ultraviolet irradiation during this period; wash with PBS buffer 3 times, 10 minutes each time, and add ultraviolet irradiation during this period; change the gel holes, add PBS / complete medium and soak for 3 days, aspirate the supernatant, wash with PBS / complete medium, and then lyophilize for standby. At this time, the free ions are basically removed completely. After sterilization and removal of free metal ions, the hydrogel is loaded with adipose-derived mesenchymal stem cells. The specific operation is as follows: Each 50 μL of hydrogel is loaded with about 10W adipose stem cells. After counting the adipose stem cells, resuspend them with 5 μL - 10 μL of complete medium. After blotting the surface moisture of the hydrogel with sterile absorbent paper, use a pipette tip to press against the surface of the gel and inject the cells.

[0051] Performance testing ① CCK8 cytotoxicity detection of gel extract After the gel was sterilized, it was soaked in complete medium for three days, and then the extract was taken. PC12 cells were cultured in three groups, namely the control group (100% complete medium), 75% extract group (75% extract + 25% complete medium), and 100% extract group (100% extract). After 24 h and 72 h of culture, the medium was aspirated and discarded, washed with PBS buffer, and then CCK8 was added, and the absorbance value at 450 nm was measured with an enzyme-labeled instrument.

[0052] ② Fluorescence images of live and dead cell staining of gel extract on ADSC cells After loading adipose-derived stem cells, fluorescence images of live and dead cell staining were taken after 1 and 3 days of culture.

[0053] The biocompatibility of the conductive hydrogel obtained in Example 3 is as Figure 4 、 5 shown. PC12 cells and adipose-derived stem cells were cultured with the hydrogel extract. Figure 4 The CCK8 results showed that the conductive hydrogel obtained in Example 3 had no cytotoxicity to PC12 cells, and subsequent in vivo applications in rats and loading of other drugs and cells could be carried out safely (According to "GB / T 16886.5 - 2017 Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity", when the reduction in cell viability exceeds 30%, a cytotoxic reaction is determined; when the reduction in cell viability is greater than 70%, it can be determined as no cytotoxicity). Figure 5 The results of live and dead staining showed that the conductive hydrogel obtained in the present invention had no cytotoxicity to adipose-derived stem cells, indicating good biocompatibility of the hydrogel of the present invention.

[0054] To evaluate the in vivo effect of the hydrogel of the present invention, immunofluorescence staining technology was used to evaluate the nerve survival and regeneration in the spinal cord tissues of rats in the spinal cord injury blank control group ( Figure 6 recorded as the spinal cord injury group in ), and the group treated with the hydrogel after spinal cord injury (using the hydrogel prepared in Example 1 of the present invention, Figure 6 recorded as the spinal cord injury / hydrogel group in ) at 6 weeks after surgery. The expression level of neurofilament (NF) and the distribution of newborn neurons (Tuj-1 positive) and mature neurons (MAP-2 positive) were mainly detected. The staining results of neuron markers showed that compared with the spinal cord injury blank control group, the number of newborn neurons (Tuj-1 positive) and mature neurons (MAP-2 positive) in the injury area of the group treated with the hydrogel after spinal cord injury using the hydrogel of the present invention increased significantly, suggesting that the treatment strategy of the present invention can effectively promote the survival and regeneration of neurons ( Figure 6A in the middle). The results of neurofilament protein staining showed that the expression of neurofilament protein in the injured part of the spinal cord injury blank control group was significantly reduced, while the conductive hydrogel transplantation of the present invention can significantly improve the loss of neurofilaments ( Figure 6 Middle B). Nissl staining results are consistent with neurofilaments ( Figure 6 The above results jointly demonstrate the significant effect of the present invention on neuron survival and nerve fiber regeneration in rats with spinal cord injury.

[0055] In recent years, stem cell therapy, as a new treatment strategy with broad application prospects, has provided a new direction for the treatment of spinal cord injury (SCI). Among them, adipose-derived stem cells (ADSCs) have attracted much attention due to their unique biological characteristics. ADSCs can be obtained from adipose tissue in a minimally invasive manner, with significant advantages such as wide sources, convenient collection, and little damage to the donor. In addition, ADSCs also show low immunogenicity and multidirectional differentiation potential, making them a research hotspot in the field of regenerative medicine. Studies have shown that under specific induction conditions, ADSCs can differentiate into neuron-like cells and glial cells, thereby replacing damaged nerve cells; at the same time, ADSCs can also release a variety of neurotrophic factors through paracrine mechanisms, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and insulin-like growth factor (IGF). These factors can not only promote the survival, proliferation and differentiation of nerve cells, but also inhibit inflammatory responses, improve the local microenvironment of injury, and create favorable conditions for nerve repair. Although ADSCs have significant therapeutic potential in theory, they still face many challenges in practical applications. First, the survival rate of cells after transplantation is low, which is mainly attributed to the complex microenvironment in the body, including immune rejection, insufficient nutrient supply, and oxidative stress, which makes it difficult for a large number of transplanted cells to survive for a long time. Secondly, transplanted cells are easily lost from the site of injury, making it difficult to achieve effective colonization and long-term residence, thereby limiting their therapeutic effect. In addition, in the absence of a suitable inducing microenvironment and supporting conditions, the efficiency of ADSCs differentiation into neural cells is low, which further affects the performance of its repair function. The hydrogel in the present invention, as a polymer material with a three-dimensional porous structure, has excellent biocompatibility, degradable properties and good plasticity, and can highly simulate the structure and function of the extracellular matrix, creating an ideal microenvironment for the adhesion, proliferation and differentiation of adipose stem cells.

[0056] 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 protection scope of the present invention.

Claims

1. A method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells, characterized in that, It includes the following steps: Step S1: After oxidizing hyaluronic acid, graft dopamine onto the oxidized hyaluronic acid to obtain oxidized hyaluronic acid-dopamine; Step S2: Aminate gelatin to obtain aminated gelatin; Step S3: Mix the oxidized hyaluronic acid-dopamine in Step S1 with the aminated gelatin in Step S2, and perform Schiff base crosslinking to obtain a hydrogel; Step S4: Load adipose mesenchymal stem cells into the hydrogel.

2. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 1, characterized in that, In the said Step S1, the specific steps of oxidizing hyaluronic acid include: Add hyaluronic acid into deionized water, stir until completely dissolved, then add sodium periodate and continue stirring, and finally add ethylene glycol to terminate the reaction. After dialysis, freeze-dry to obtain oxidized hyaluronic acid; The mass ratio of the hyaluronic acid, sodium periodate, ethylene glycol to deionized water is: 0.2 - 0.3:0.10 - 0.15:0.10 - 0.15:2 - 3.

3. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 2, characterized in that, In the said Step S1, the specific steps of grafting dopamine include: Dissolve the oxidized hyaluronic acid in deionized water, adjust the pH to 5 - 6, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and stir for activation; add dopamine hydrochloride, stir and react for 24h - 30h, and after dialysis, freeze-dry to obtain oxidized hyaluronic acid-dopamine; The mass ratio of the oxidized hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide to dopamine hydrochloride is: 8 - 10:6.6 - 7.0:1.95 - 2.00:4 - 5.

4. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 3, characterized in that, In the said Step S1, the oxidation treatment of hyaluronic acid is carried out under dark conditions, and the reaction time is 4h - 5h; the dopamine grafting reaction is carried out under N2 atmosphere.

5. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 1, characterized in that, In the said Step S2, the specific steps of aminating gelatin include: Dissolve gelatin in PBS buffer, disperse evenly, add ethylenediamine, adjust the pH to 5 - 6, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to start the reaction. After the reaction ends, dialyze and then freeze-dry for standby; The mass ratio of the gelatin, PBS buffer, ethylenediamine to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is: 0.5 - 1.0:10 - 20:0.8 - 1.0:0.23 - 0.

30.

6. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 5, characterized in that, In Step S2, the concentration of the PBS buffer is 0.1M - 0.2M. Gelatin needs to be heated and dispersed when dissolved in the PBS buffer. The temperature of the gelatin amination reaction is 35°C - 40°C, and the reaction time is 24h - 30h.

7. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 1, characterized in that, In the said Step S3, use ferric chloride as a crosslinking agent to coordinate with catechol to form a gel; the mixing volume ratio of the oxidized hyaluronic acid-dopamine to the aminated gelatin is 10:1 - 20:1, the concentration of ferric chloride is 2.5mM - 3mM, and the addition amount is 10% - 20% of the system.

8. The method for preparing a conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 1, characterized in that, In the said Step S4, the loading of adipose mesenchymal stem cells is carried out after sterilization and treatment for removing free metal ions.

9. A conductive hydrogel loaded with adipose mesenchymal stem cells prepared by the preparation method according to any one of claims 1 to 8.

10. An application of the conductive hydrogel loaded with adipose mesenchymal stem cells according to claim 9, characterized in that, For preparing drugs or scaffolds for nerve tissue regeneration therapy.

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