A titanium carbide-metal ion conductive hydrogel with biological activity and a preparation method and application thereof

Abstract

The application belongs to the technical field of spinal cord injury repair, and particularly relates to a biologically active titanium carbide-metal ion conductive hydrogel as well as a preparation method and application thereof. The biologically active titanium carbide-metal ion conductive hydrogel is formed by dynamic covalent bond crosslinking of two-dimensional titanium carbide-metal ion composite nanosheets, aldehyde-based hyaluronic acid and amino-based gelatin. The conductive hydrogel prepared by the application can effectively remove excess ROS at an injury site, reduce neuron oxidative stress, effectively promote axon regeneration, myelin sheath formation and neural network reconstruction, and thus accelerate recovery of motor function.

Description

Technical Field

[0001] This invention relates to a bioactive titanium carbide-metal ion conductive hydrogel, its preparation method and application, belonging to the field of spinal cord injury repair technology. Background Technology

[0002] Spinal cord injury is a highly prevalent and disabling injury to the central nervous system, potentially causing devastating, lifelong disability. A series of pathological processes following this injury limit axonal regeneration, including increased neuronal death, mitochondrial dysfunction, and the formation of an inhibitory reactive oxygen species (ROS) microenvironment around the lesion. Current clinical treatments still have limitations in effectively regulating mitochondrial function, improving neuronal survival, and promoting axonal regeneration and functional synaptic connections.

[0003] Tissue repair strategies based on biomaterial scaffolds are considered promising, as they can modulate the intrinsic regenerative capacity of neurons and improve the inhibitory microenvironment by providing suitable topological structures, biophysical and biochemical signals, thereby promoting spinal cord injury repair. Among these, conductive hydrogels, by mimicking the electrophysiological properties of neural tissue, can provide a dynamic electrical microenvironment for axonal regeneration. In recent years, two-dimensional transition metal carbide, nitride, and carbonitride materials (MXene materials) have attracted attention due to their excellent conductivity, hydrophilicity, biodegradability, and biocompatibility. Conductive hydrogels based on these MXene materials provide a promising platform for spinal cord injury repair.

[0004] However, the application of such materials in spinal cord injury repair still faces challenges. First, MXene components exhibit poor stability in the physiological environment, potentially affecting their long-term ability to maintain electrical signal transmission and support axonal regeneration. Second, traditional MXene conductive hydrogels have limited effectiveness in regulating the microenvironment of excessive reactive oxygen species in the injury area, usually requiring the introduction of other functional components. This often leads to system complexity and may affect the overall effectiveness and reliability of the treatment. Therefore, how to enhance the bioactivity and microenvironment regulation capabilities of hydrogels while maintaining their conductivity through rational material modification and design remains a problem that needs to be solved. Summary of the Invention

[0005] This invention addresses the aforementioned technical problems of the prior art by providing a bioactive titanium carbide-metal ion conductive hydrogel, its preparation method, and its applications. Two-dimensional titanium carbide-metal ion composite nanosheets are prepared via a solvothermal method. These nanosheets are then co-assembled with modified hyaluronic acid and gelatin using a blending method to achieve the preparation of an injectable hydrogel with ROS scavenging and conductive functions. This hydrogel shows promising application prospects in the field of spinal cord injury repair and provides broader insights for the development of injectable spinal cord tissue engineering materials.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] One of the objectives of this invention is to provide a bioactive titanium carbide-metal ion conductive hydrogel, which is formed by cross-linking two-dimensional titanium carbide-metal ion composite nanosheets, aldehyde-based hyaluronic acid, and amino-based gelatin through dynamic covalent bonds.

[0008] Furthermore, in the two-dimensional titanium carbide-metal ion composite nanosheets, the metal ion is Ce. 3+ The two-dimensional titanium carbide-metal ion composite nanosheets are surface-modified with Ce(OH) ions. x Layer, where 0 < x < 3.

[0009] Furthermore, the mass ratio of each raw material component is as follows: 1-5 parts of two-dimensional titanium carbide-metal ion composite nanosheets, 30-50 parts of aldehyde-modified hyaluronic acid, and 100-150 parts of amino-modified gelatin.

[0010] A second objective of this invention is to provide a method for preparing the above-mentioned titanium carbide-metal ion conductive hydrogel, comprising the following steps:

[0011] S1. Preparation of two-dimensional titanium carbide-metal ion composite nanosheets;

[0012] S2. Prepare aldehyde-modified hyaluronic acid and amino-modified gelatin respectively;

[0013] S3. Preparation of two-dimensional titanium carbide-metal ion composite nanosheet dispersion;

[0014] S4. Dissolve the aldehyde-modified hyaluronic acid obtained in step S2 in the dispersion containing two-dimensional titanium carbide-metal ion composite nanosheets obtained in step S3 to obtain solution A; dissolve the aminated gelatin obtained in step S2 in a buffer solution to obtain solution B.

[0015] S5. Mix solution A obtained in step S4 with solution B and stir to obtain titanium carbide-metal ion conductive hydrogel.

[0016] Furthermore, step S1 specifically includes the following steps:

[0017] S11. Disperse multilayer titanium carbide nanosheets in water and deoxygenate them.

[0018] S12. Under an inert atmosphere, the dispersion obtained in step S11 is subjected to ultrasonic treatment, followed by centrifugation and collection of the supernatant to obtain a titanium carbide nanosheet dispersion.

[0019] S13. Mix the titanium carbide nanosheet dispersion obtained in step S12 with the cerium salt solution and stir at room temperature to carry out the ion recombination reaction.

[0020] S14. The mixture after the reaction in step S13 is centrifuged and washed to obtain the two-dimensional titanium carbide-metal ion composite nanosheets.

[0021] Furthermore, in step S12, the cerium salt is cerium nitrate, and the mass ratio of cerium ions to titanium carbide nanosheets in the cerium salt solution is 0.01 to 0.02: 1.

[0022] Furthermore, in step S2,

[0023] The preparation of aminated gelatin includes the following steps:

[0024] (1) In the presence of a condensing agent, a gelatin solution is mixed with a compound containing an acylhydrazine group, so that the carboxyl groups on the gelatin molecular chain undergo an amidation reaction with the compound containing the acylhydrazine group, thereby introducing an amino group;

[0025] (2) The reaction product was purified to obtain the aminated gelatin.

[0026] Furthermore, the compound containing the hydrazide group is adipic acid dihydrazide, the condensing agent includes carbodiimide condensing agents and hydroxybenzotriazole activators, the mass ratio of the compound containing the hydrazide group to the gelatin is 4-5:1, and the mass ratio of the condensing agent to the gelatin is 0.4-0.5:1.

[0027] Furthermore, the carbodiimide condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the hydroxybenzotriazole activator is 1-hydroxybenzotriazole (HOBt).

[0028] Furthermore, in step S2,

[0029] The preparation of aldehyde-modified hyaluronic acid includes the following steps:

[0030] (1) Dissolve hyaluronic acid in a first solvent to obtain a hyaluronic acid solution; dissolve the oxidant in a second solvent to obtain an oxidant solution;

[0031] (2) Under stirring and light-protected conditions, the oxidant solution and the hyaluronic acid solution are mixed to carry out an oxidation reaction, so as to introduce aldehyde groups on the hyaluronic acid molecular chain;

[0032] (3) After the reaction is completed, a quencher is added to terminate the reaction, and the reaction product is purified to obtain the aldehyde-modified hyaluronic acid.

[0033] Furthermore, the oxidant is periodate, the mass ratio of the oxidant to the hyaluronic acid is 0.3 to 0.35:1, and the quenching agent is one of ethylene glycol, glycerol, or sodium sulfite.

[0034] A third objective of this invention is to provide the application of the above-mentioned bioactive titanium carbide-metal ion conductive hydrogel in the preparation of medical materials for repairing nerve damage.

[0035] Furthermore, the nerve injury is a spinal cord injury, and the medical material is an injectable tissue-engineered scaffold.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] I. This invention utilizes Ce 3+ In-situ modification was performed to form Ce(OH) on the surface of titanium carbide nanosheets. x The layers work together to promote interlayer electron transfer, resulting in two-dimensional titanium carbide-metal ion composite nanosheets that not only maintain the inherent high conductivity of MXene materials but also further enhance its conductivity. More importantly, they effectively isolate the nanosheets from the erosion of water and oxygen, significantly improving the material's antioxidant stability. Simultaneously, they endow the nanosheets with excellent ROS scavenging capabilities, solving the key problem of easy oxidation and degradation of traditional MXene materials in physiological environments.

[0038] II. This invention introduces two-dimensional titanium carbide-metal ion composite nanosheets with dual functions of conductivity and anti-oxidation into a hydrogel network. Through physical blending and chemical cross-linking, a smart biomaterial integrating conductivity, anti-oxidation, injectability, and self-healing is obtained without the need to add other complex components.

[0039] Third, the hydrogel prepared by this invention has both good injectability and outstanding self-healing properties. Injectability allows it to precisely fill irregular tissue damage areas through minimally invasive methods; self-healing ability ensures that the material maintains structural integrity after implantation, can adapt to the dynamic mechanical environment in the body, and provides long-term stable three-dimensional support for cells.

[0040] IV. The conductive hydrogel prepared by this invention can effectively remove excess ROS at the site of injury and reduce neuronal oxidative stress; its conductivity helps to rebuild the electrophysiological microenvironment, promote neuronal adhesion, axonal directional growth and mitochondrial function recovery; animal experiments show that when this material is implanted as an injectable scaffold, it can effectively promote axonal regeneration, myelin formation and neural network reconstruction, thereby accelerating the recovery of motor function. Attached Figure Description

[0041] Figure 1 Scanning electron microscope (SEM) image of the two-dimensional titanium carbide-metal ion composite nanosheets prepared for an embodiment of the present invention.

[0042] Figure 2 Transmission electron microscope (TEM) image of the two-dimensional titanium carbide-metal ion composite nanosheets prepared for an embodiment of the present invention.

[0043] Figure 3 This is a comparison of the electrical conductivity of titanium carbide nanosheets and two-dimensional titanium carbide-metal ion composite nanosheets.

[0044] Figure 4 This is a comparison of the degradation capabilities of titanium carbide nanosheets and two-dimensional titanium carbide-metal ion composite nanosheets for hydrogen peroxide.

[0045] Figure 5 Comparison of TEM images (a) and X-ray diffraction patterns (b) of titanium carbide nanosheets and two-dimensional titanium carbide-metal ion composite nanosheets after being placed in air for 3 weeks.

[0046] Figure 6 Photographs of the injectable process of the conductive hydrogel prepared according to an embodiment of the present invention.

[0047] Figure 7 A demonstration photograph of the conductive hydrogel used to light up an LED circuit, prepared according to an embodiment of the present invention.

[0048] Figure 8 Fluorescence intensity map showing the effect of different treatment groups on intracellular ROS levels in neurons (DCFH-DA staining).

[0049] Figure 9 Fluorescence intensity maps showing the effects of different treatment groups on neuronal axonal mitochondrial transport (TOMM20 and β-III Tubulin staining).

[0050] Figure 10 In vivo fluorescence staining images (DHE staining) of ROS levels in the damaged areas of each treatment group.

[0051] Figure 11 Fluorescence intensity maps of axonal regeneration (β-III Tubulin) and mitochondrial distribution (TOMM20) in the damaged areas of each treatment group.

[0052] Figure 12 Fluorescence intensity maps of axons (β-III Tubulin) and myelin sheaths (MBP) in the damaged areas of each treatment group. Detailed Implementation

[0053] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0054] Example

[0055] I. Preparation of Two-Dimensional Titanium Carbide-Metal Ion Composite Nanosheets

[0056] a. Slowly add 0.5 g of lithium fluoride (LiF) to 10 mL of 9 M hydrochloric acid (HCl) solution and stir until dissolved. Then add 0.5 g of Ti3AlC2 powder and stir for 20 min.

[0057] b. Transfer to a stainless steel reactor and react at 60°C for 24 h. After the reaction is complete, cool to room temperature, wash the product 6 times with deionized water and once with ethanol, and dry the product overnight in a vacuum drying oven to obtain multilayer titanium carbide nanosheets (m-Ti3C2).

[0058] c. Disperse 100 mg of m-Ti3C2 in 10 mL of deionized water and purge with argon (Ar) gas for 15 min to remove oxygen. Then, sonicate the dispersion for 1 h under argon protection.

[0059] d. Centrifuge the above dispersion at 3500 rpm for 1 h, collect the supernatant, and obtain Ti3C2T. x The dispersion had a concentration of 10 mg / mL.

[0060] e. Take 10 mL of the above Ti3C2T x The dispersion (10 mg / mL) was diluted with 90 mL of deionized water. While stirring, 30 μL of a 40 mM Ce(NO3)3·6H2O aqueous solution was slowly added, and the reaction was continued at room temperature for 6 h with stirring.

[0061] f. Centrifuge the above reaction solution at 11000 rpm for 10 min, collect the precipitate, and wash it three times with deionized water to obtain two-dimensional titanium carbide-metal ion composite nanosheets (Ti3C2T). x -Ce 3+ (Composite nanosheets).

[0062] SEM and TEM images of the two-dimensional titanium carbide-metal ion composite nanosheets prepared above are shown below. Figure 1 and 2 As shown, the composite nanosheets obtained in the embodiments of the present invention exhibit a typical two-dimensional sheet-like structure with uniform size, approximately 200 nm. This composite heterostructure facilitates electrolyte ion transport and significantly enhances electronic conductivity and antioxidant properties.

[0063] II. Ti3C2T x -Ce 3+ Performance characterization of composite nanosheets

[0064] 1. Conductivity test:

[0065] Titanium carbide nanosheets (pure Ti3C2T) xThe dispersion and the two-dimensional titanium carbide-metal ion composite nanosheets (Ti3C2T) obtained in the embodiments of the present invention x -Ce 3+ The composite nanosheet dispersions were filtered and naturally dried to obtain self-supporting films, and their conductivity was measured using a four-probe tester.

[0066] The results are as follows Figure 3 As shown, Ti3C2T x -Ce 3+ The electrical conductivity of the composite nanosheet film reached 4433 S / cm, which is significantly higher than that of pure Ti3C2T. x The membrane's S / cm is 3152. This is attributed to Ce. 3+ The ionic cross-linking network formed between ions and the terminal groups on the surface of titanium carbide nanosheets facilitates interlayer electron transfer. Furthermore, the Ce(OH) groups formed on the surface of the titanium carbide nanosheets... x The base shielding layer acts as an active bridge, promoting interlayer charge transfer and enhancing the conductivity of titanium carbide-Ce. 3+ Electronic coupling within the framework.

[0067] 2. ROS removal performance test:

[0068] Take 0.2 mL of each of the two nanosheet dispersions with a concentration of 5 mg / mL and place them in 5 mL of H2O2 solution with a concentration of 100 µM. After standing for 9 h, perform a hydrogen peroxide degradation test to determine the concentration of H2O2 solution.

[0069] The results are as follows Figure 4 As shown, Ti3C2T x -Ce 3+ The composite nanosheets exhibited significantly enhanced H2O2 degradation efficiency after 9 hours, reaching 76.7%, which is far higher than that of pure Ti3C2T. x The nanosheets account for 38.2%, twice that of titanium carbide nanosheets. This is attributed to Ce(OH)₂. x The unique shielding layer of the hydroxyl complex, through Ce 3+ / Ce 4+ Redox cycling effectively enhanced ROS scavenging capacity and maintained the structure of titanium carbide. Furthermore, Ce(OH) x As a protective redox active layer, the layer can preferentially oxidize to generate CeO2, thereby maintaining the two-dimensional layered structure and passing through Ce. 3+ / Ce 4+ Redox cycles enhance ROS scavenging ability, which is beneficial for improving antioxidant performance and structural stability.

[0070] 3. Antioxidant stability test:

[0071] Ti3C2T x Dispersion of nanosheets (10 mg / mL), Ti3C2T x -Ce 3+ The dispersion of the composite nanosheets (10 mg / mL) was placed in 20 mL glass bottles and allowed to stand for 3 weeks. The morphological and structural changes were observed by TEM and XRD.

[0072] The results are as follows Figure 5 As shown, where Figure 5 In the middle, 'a' represents a TEM image. Figure 5 In the image, b represents the XRD pattern of Ti3C2T. x The nanosheets exhibited significant oxidative degradation, with spindle-shaped and rod-shaped TiO2 nanorods appearing on the surface and edges. The characteristic XRD peak at 6.3° disappeared, while a distinct TiO2 diffraction peak appeared at 25.3°, indicating that Ti3C2T... x The nanosheets underwent severe structural degradation and oxidation; while Ti3C2T x -Ce 3+ The composite nanosheets retain their intact two-dimensional sheet structure, indicating excellent morphological stability. The XRD characteristic peak at 6.3° is clear, and the characteristic peak of CeO2 only appears at 28.3°, proving that Ce(OH)2 is present. x The layer effectively protects Ti3C2T x The structure maintains the structural stability of titanium carbide. These results indicate that Ce 3+ The embedding effectively isolates the titanium carbide nanosheets from water molecules and dissolved oxygen, thereby enhancing the antioxidant properties of the titanium carbide nanosheets.

[0073] III. Preparation of Aldehyde-CHO

[0074] (1) Weigh 1.0 g of sodium hyaluronate (HA) and dissolve it in 90 mL of deionized water. Weigh 321 mg of sodium periodate (NaIO4) and dissolve it in 10 mL of deionized water.

[0075] (2) Under stirring conditions, NaIO4 solution was added dropwise to HA solution at room temperature (25°C). o React under light-protected conditions at C for 48 hours.

[0076] (3) After the reaction was complete, 500 µL of ethylene glycol was added to quench the reaction. The reaction solution was placed in a dialysis bag (molecular weight cutoff of 30,000) and dialyzed in deionized water for 3 days, with the water changed every 8 hours. Finally, the product was freeze-dried to obtain HA-CHO solid. The product was placed in a 4-inch glass container. o Store in refrigerator C.

[0077] IV. Preparation of Aminated Gelatin (Gel-ADH)

[0078] (1) Weigh 4.0 g of gelatin and dissolve it in 600 mL of deionized water at 50 °C. Weigh 16.7 g of adipic acid dihydrazide (ADH) and add it to the above solution, stirring to dissolve. Separately, dissolve 1.92 g of EDC and 1.35 g of HOBt in 10 mL of a mixed solvent of DMSO and deionized water (v:v=1:1), and use sonication to aid dissolution. Under stirring, slowly add the EDC / HOBt mixed solution to the gelatin / ADH mixed solution and react at room temperature for 24 h.

[0079] (2) After the reaction was complete, the solution was placed in a dialysis bag (molecular weight cutoff of 30,000) and dialyzed in deionized water for 3 days, changing the water every 8 hours. Finally, the product was freeze-dried to obtain Gel-ADH solid. The product was placed in a 4-degree Celsius environment. o Store in refrigerator C.

[0080] V. Preparation of bioactive titanium carbide-metal ion conductive hydrogels

[0081] a. Weigh 40 mg of the HA-CHO prepared above and dissolve it in a solution containing 0.02 mL Ti3C2T x -Ce 3+ The composite nanosheet dispersion (10 mg / mL) was vortexed in 2 mL of PBS buffer until completely dissolved to obtain solution A.

[0082] b. Weigh 120 mg of the prepared Gel-ADH and dissolve it in 2 mL of PBS buffer until it is evenly dispersed to obtain solution B.

[0083] c. Take 200 µL of solution A and solution B respectively, mix and stir for 30 s to obtain bioactive titanium carbide-metal ion conductive hydrogel.

[0084] like Figure 6 As shown, the mixed liquid rapidly gels within approximately 30 seconds, forming a black, homogeneous hydrogel that can be easily extruded through a syringe to form continuous strips, demonstrating its good injectability.

[0085] Connect the resulting conductive hydrogel to the circuit, such as Figure 7 As shown, the LED light can be successfully lit, proving that it has good conductivity.

[0086] VI. Regulatory effect of composite nanosheets on neuronal oxidative stress (in vitro)

[0087] Neurons were extracted and cultured from newborn SD rats.

[0088] The experiment was divided into three groups: control group (normal culture), peroxidation group (treated with H2O2), and treatment group (treated with H2O2 and Ti3C2T prepared in step one of this invention). x -Ce 3+ (Composite nanosheets).

[0089] After treatment with 3% hydrogen peroxide (final concentration 100 μmol / L) for 24 h, the total ROS level in cells was detected using a reactive oxygen species fluorescent probe (DCFH-DA probe). The DCFH-DA probe was diluted with serum-free medium at a ratio of 1:1000; the original medium in the cell culture plate was removed, and 500 μL of the diluted probe was added to each well. The plate was then incubated at 37 ℃ in the dark for 30 min. The supernatant in the wells was discarded, and the cells were washed twice with preheated serum-free medium to remove any probe that had not entered the cells. 1× Hoechst 33342 nuclear staining solution was added, and the nuclei were stained for 10 min to label the nuclei. The cells were then washed once with preheated serum-free medium, replaced with complete medium, and the fluorescence expression intensity was observed under a fluorescence microscope.

[0090] The results are as follows Figure 8 As shown, the fluorescence intensity in the peroxidation group was significantly enhanced, indicating an increased level of oxidative stress, which led to a significant increase in total ROS levels and resulted in impaired mitochondrial function and increased superoxide anion content. Compared with the peroxidation group, the fluorescence intensity in the treatment group was significantly reduced, even approaching that of the control group, indicating that the Ti3C2T of the present invention... x -Ce 3+ Composite nanosheets can effectively remove ROS and alleviate oxidative stress in neuronal cells.

[0091] VII. Composite nanosheets promote neuronal axon regeneration (in vitro)

[0092] The TOMM20 and β-III Tubulin antibodies were used to assess mitochondrial transport in neuronal axons. TOMM20 is a core component of the mitochondrial outer membrane transporter complex (TOM complex) and a marker of mitochondrial dysfunction. β-III Tubulin is an antibody that specifically recognizes neuronal β-III tubulin and is commonly used in medical research and pathological diagnosis of mature neuronal states.

[0093] The experiment was divided into three groups: control group (normal culture), peroxidation group (treated with H2O2), and treatment group (treated with H2O2 and the Ti3C2T of this invention). x -Ce 3+(Composite nanosheets). Hydrogen peroxide was added to the culture medium of both the peroxide group and the treatment group for 24 hours. The supernatant was then discarded, and an appropriate amount of preheated serum-free culture medium was added. Excess hydrogen peroxide was removed by gentle shaking. Appropriate amounts of culture medium were then added to the cells in each of the three groups, with Ti3C2T added to the treatment group. x -Ce 3+ The composite nanosheets were concentrated to a concentration of 40 μg / mL and cultured for another 24 h. The well plates were removed, the supernatant discarded, and cells were fixed with 4 wt% paraformaldehyde for 30 min. The supernatant was discarded again, and the cells were washed three times with PBS, 5 min each time. Then, antibodies against TOMM20 and β-III Tubulin diluted in blocking buffer (10 vt% goat serum + 0.1 vt% Triton + PBS) were added, and the plates were incubated overnight at 4 °C. The next day, the well plates were removed, the supernatant discarded, and the cells were washed three times with PBS buffer, 5 min each time. Then, secondary antibody diluted in PBS was added, and the plates were incubated at room temperature for 1 h. The supernatant was discarded again, and the cells were washed three times with PBS buffer, 5 min each time. The smears were carefully removed and placed on a glass slide. An appropriate amount of anti-fluorescence attenuation mounting medium containing 4',6-diamidinyl-2-phenylindole (DAPI) was added, and the smears were inverted onto the mounting medium for imaging and observation using laser confocal microscopy.

[0094] The results are as follows Figure 9 As shown, in the peroxidation group, neuronal axons atrophied and broke, and mitochondria were fragmented. In contrast, the treatment group showed significantly improved neuronal axon length and branching complexity, and mitochondria were continuously and uniformly distributed along the axons, demonstrating the effectiveness of the Ti3C2T method of this invention. x -Ce 3+ Composite nanosheets effectively promote axon growth and normal mitochondrial transport by regulating the inhibitory ROS microenvironment and maintaining electrical signal transmission. They also promote the influx of calcium ions into axons and mitochondria by activating calcium channels (electrical signals activate voltage-gated calcium channels by changing the axon membrane potential, promoting the influx of calcium ions and their specific uptake by mitochondria, and driving axon growth and normal mitochondrial transport through energy supply and molecular signal regulation), thereby promoting neuronal axon regeneration.

[0095] 8. Conductive hydrogel promotes spinal cord injury repair in rats (in vivo)

[0096] Animal model establishment and treatment: Sprague-Dawley rats (SD rats) were randomly divided into three groups: injury group (spinal cord injury only), hydrogel group (injection of blank hydrogel after injury), and treatment group (injection of Ti3C2T of this invention after injury). x -Ce 3+(Conductive hydrogel). Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg). After the corneal reflex disappeared, a 3 cm incision was made centered on the T9-T11 segment. The fascia was cut, and the muscles above the spinous process were removed. The spinous process was then removed using mosquito forceps, fully exposing the spinal cord. Next, approximately 0.5 cm of spinal cord tissue was removed at T9 using a No. 7 scalpel. Immediately after transection, hind limb twitching and tail flicking were observed, followed by the loss of tension in the hind limbs and tail. After hemostasis, the hydrogel group and the treatment group were injected with blank hydrogel and the Ti3C2T of the present invention, respectively. x -Ce 3+ Conductive hydrogels are injected into the injury cavity. The injured area is left untreated. Then, the muscles, fascia and skin are sutured layer by layer to form a complete spinal cord transection injury model.

[0097] Oxidative stress level assessment: Eight weeks after spinal cord injury, frozen sections of the injured spinal cord were obtained and stained with DHE probes. The specific steps were as follows: Frozen sections were thawed, immersed in PBS to remove embedding medium, and the tissue area was delineated with a histochemical pen. Antibody of DHE diluted with blocking buffer was added to the tissue, and the tissue was incubated overnight at 4 °C. The supernatant was discarded, and the tissue was washed three times with PBS (5 min each time). Secondary antibody diluted with PBS was added, and the tissue was incubated at room temperature for 1 h. The supernatant was discarded, and the tissue was washed three times with PBS. Finally, a mounting medium containing DAPI was added, and the tissue was covered with a coverslip. The tissue was observed and photographed under a fluorescence microscope to detect the superoxide anion level. Results are as follows: Figure 10 As shown, the damaged group exhibited strong red fluorescence, indicating that secondary injury-induced mitochondrial dysfunction and inflammatory response exacerbated oxidative stress and neuronal damage. The average fluorescence intensity of the hydrogel group was slightly lower, indicating that the antioxidant effect of the blank hydrogel was limited. Notably, the fluorescence intensity of the treatment group was the weakest, significantly lower than that of the damaged group and the hydrogel group, demonstrating the effectiveness of the Ti3C2T of this invention. x -Ce 3+ Conductive hydrogels can effectively remove excess ROS in vivo and improve the local microenvironment of the damaged area, thereby creating a favorable microenvironment for neuronal survival and axonal regeneration.

[0098] Axonal regeneration and mitochondrial function assessment: Spinal cord tissue sections were subjected to immunofluorescence double labeling with β-III Tubulin and TOMM20. The specific steps were as follows: Frozen sections were thawed, immersed in PBS to remove the embedding agent, and the tissue area was delineated using a histochemical pen. Antibodies of TOMM20 and β-III Tubulin diluted with blocking buffer were added to the tissue and incubated overnight at 4 °C. The supernatant was discarded, and the tissue was washed three times with PBS for 5 min each time. Secondary antibody diluted with PBS was added, and the tissue was incubated at room temperature for 1 h. The supernatant was discarded, and the tissue was washed three times with PBS. Finally, anti-fluorescence attenuation mounting medium containing DAPI was added, a coverslip was placed, and the tissue was observed and photographed using a fluorescence microscope.

[0099] The results are as follows Figure 11 As shown, the damaged group exhibited only sparse β-III tubulin and discontinuous TOMM20 distribution, indicating axonal structural damage and impaired mitochondrial transport. In the hydrogel group, some improvement was observed compared to the damaged group, but the overall repair effect remained limited. Specifically, the number of β-III-tubulin-positive axons increased compared to the damaged group, and axonal continuity was partially restored, suggesting that the hydrogel scaffold provided physical support and a favorable regenerative microenvironment for axonal growth to some extent. However, the overall arrangement of these regenerated axons remained disordered, and the axonal connections across the damaged area were not dense and orderly, failing to form a continuous and complete axonal bundle structure. This indicates that the hydrogel mainly functions by providing structural support and buffering the damaged microenvironment, but lacks the ability to actively regulate mitochondrial function and axonal energy metabolism, making it difficult to achieve high-level axonal regeneration and neural network reconstruction. Compared to the damaged group, the treated group showed a large number of continuous β-III-tubulin axons and a uniform and elongated mitochondrial distribution along the regenerated axons, demonstrating the superiority of the Ti3C2T scaffold of this invention. x -Ce 3+ Conductive hydrogels can promote the maintenance and transport of mitochondria, thereby promoting axon regeneration and neural network reconstruction.

[0100] Myelin regeneration assessment: Spinal cord tissue sections were subjected to immunofluorescence double labeling with β-III Tubulin and myelin basic protein (MBP). The specific steps were as follows: Frozen sections were thawed, immersed in PBS to remove the embedding agent, and the tissue area was delineated using a histochemical pen. Antibodies diluted with blocking buffer for MBP and β-III Tubulin were added to the tissue, and the sections were incubated overnight at 4 °C. The supernatant was discarded, and the sections were washed three times with PBS for 5 min each time. Secondary antibody diluted with PBS was added, and the sections were incubated at room temperature for 1 h. The supernatant was discarded, and the sections were washed three times with PBS. A mounting medium containing DAPI was added, and the sections were covered with coverslips. The sections were observed and photographed under a fluorescence microscope. The results are as follows: Figure 12As shown, the injury group exhibited sparse and disordered β-III tubulin fibers and significantly reduced MBP expression, which together indicate impaired neuronal axonal integrity and widespread demyelination. The hydrogel group showed a slight increase in β-III tubulin and MBP-positive regions, suggesting that the hydrogel itself has limited biological regulatory effect on neuronal and myelin regeneration. In the treatment group, the proportion of β-III tubulin-positive axons was significantly increased, and the proportion of continuous MBP-positive myelin sheaths was also significantly enhanced, indicating that Ti3C2T… x -Ce 3+ Conductive hydrogels can promote neuronal axon regeneration and effective myelin formation. More importantly, in the treatment group, a large number of regenerated axons were encapsulated by continuous MBP-positive myelin sheaths, indicating that Ti3C2T x -Ce 3+ Conductive hydrogels can effectively promote the reconstruction of axon-myelin structures, thereby restoring nerve signal transmission and functional recovery after spinal cord injury.

[0101] The above experimental results show that the Ti3C2T prepared by this invention... x -Ce 3+ Conductive hydrogels can effectively promote axonal regeneration, mitochondrial function recovery, and myelin formation after spinal cord injury by clearing ROS and regulating neuronal mitochondrial function, providing an ideal scaffold material for neural function reconstruction.

[0102] 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 within the protection scope of the present invention.

Claims

1. A bioactive titanium carbide-metal ion conductive hydrogel, characterized in that, It is formed by cross-linking two-dimensional titanium carbide-metal ion composite nanosheets, aldehyde-based hyaluronic acid and amino-based gelatin through dynamic covalent bonds; In the two-dimensional titanium carbide-metal ion composite nanosheets, the metal ion is Ce. 3+ The two-dimensional titanium carbide-metal ion composite nanosheets are surface-modified with Ce(OH) ions. x Layer, where 0 < x < 3.

2. The bioactive titanium carbide-metal ion conductive hydrogel according to claim 1, characterized in that, The mass ratio of each raw material component is as follows: 1-5 parts of two-dimensional titanium carbide-metal ion composite nanosheets, 30-50 parts of aldehyde-modified hyaluronic acid, and 100-150 parts of amino-modified gelatin.

3. A method for preparing a titanium carbide-metal ion conductive hydrogel as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Preparation of two-dimensional titanium carbide-metal ion composite nanosheets; S2. Prepare aldehyde-modified hyaluronic acid and amino-modified gelatin respectively; S3. Preparation of two-dimensional titanium carbide-metal ion composite nanosheet dispersion; S4. Dissolve the aldehyde-modified hyaluronic acid obtained in step S2 in the dispersion containing two-dimensional titanium carbide-metal ion composite nanosheets obtained in step S3 to obtain solution A; dissolve the aminated gelatin obtained in step S2 in a buffer solution to obtain solution B. S5. Mix solution A obtained in step S4 with solution B and stir to obtain titanium carbide-metal ion conductive hydrogel.

4. The method for preparing the bioactive titanium carbide-metal ion conductive hydrogel according to claim 3, characterized in that, Step S1 specifically includes the following steps: S11. Disperse multilayer titanium carbide nanosheets in water and deoxygenate them. S12. Under an inert atmosphere, the dispersion obtained in step S11 is subjected to ultrasonic treatment, followed by centrifugation and collection of the supernatant to obtain a titanium carbide nanosheet dispersion. S13. Mix the titanium carbide nanosheet dispersion obtained in step S12 with the cerium salt solution and stir at room temperature to carry out the ion recombination reaction. S14. The mixture after the reaction in step S13 is centrifuged and washed to obtain the two-dimensional titanium carbide-metal ion composite nanosheets.

5. The method for preparing the bioactive titanium carbide-metal ion conductive hydrogel according to claim 4, characterized in that, In step S12, the cerium salt is cerium nitrate, and the mass ratio of cerium ions to titanium carbide nanosheets in the cerium salt solution is 0.01 to 0.02:

1.

6. The method for preparing the bioactive titanium carbide-metal ion conductive hydrogel according to claim 3, characterized in that, In step S2, The preparation of aminated gelatin includes the following steps: (1) In the presence of a condensing agent, a gelatin solution is mixed with a compound containing an acylhydrazine group, so that the carboxyl groups on the gelatin molecular chain undergo an amidation reaction with the compound containing the acylhydrazine group, thereby introducing an amino group; (2) The reaction product was purified to obtain the aminated gelatin; The preparation of aldehyde-modified hyaluronic acid includes the following steps: (1) Dissolve hyaluronic acid in a first solvent to obtain a hyaluronic acid solution; dissolve the oxidant in a second solvent to obtain an oxidant solution; (2) Under stirring and light-protected conditions, the oxidant solution and the hyaluronic acid solution are mixed to carry out an oxidation reaction, so as to introduce aldehyde groups on the hyaluronic acid molecular chain; (3) After the reaction is completed, a quencher is added to terminate the reaction, and the reaction product is purified to obtain the aldehyde-modified hyaluronic acid.

7. The method for preparing the bioactive titanium carbide-metal ion conductive hydrogel according to claim 6, characterized in that, The hydrazide-containing compound is adipic acid dihydrazide, the condensing agent includes carbodiimide condensing agents and hydroxybenzotriazole activators, the mass ratio of the hydrazide-containing compound to the gelatin is 4-5:1, the mass ratio of the condensing agent to the gelatin is 0.4-0.5:1; the oxidizing agent is periodate, the mass ratio of the oxidizing agent to the hyaluronic acid is 0.3-0.35:1, and the quenching agent is one of ethylene glycol, glycerol, or sodium sulfite.

8. The application of the bioactive titanium carbide-metal ion conductive hydrogel as described in claim 1 or 2 in the preparation of medical materials for repairing nerve damage.

9. The application of the bioactive titanium carbide-metal ion conductive hydrogel according to claim 8, characterized in that, The nerve injury is a spinal cord injury, and the medical material is an injectable tissue-engineered scaffold.

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