Hydrogel scaffold material for spinal cord injury repair as well as preparation method and application of hydrogel scaffold material

Abstract

The invention relates to the technical field of biological materials, in particular to a hydrogel scaffold material for spinal cord injury repair as well as a preparation method and application of the hydrogel scaffold material. The hydrogel scaffold material AP39 (AP39-coated LIP) for spinal cord injury repair is prepared by combining the liposome-coated AP39 (AP39-coated LIP) serving as a drug with a GelMA hydrogel scaffold, and a scaffold is provided for injured spinal cords, so that axon regeneration of injured parts is guided, mitochondria of the injured parts is targeted to release H2S, neuronal injury is relieved, and the spinal cord injury repair effect is improved. Therefore, nerve regeneration and functional recovery after spinal cord injury are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a hydrogel scaffold material for repairing spinal cord injury and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Spinal cord injury (SCI) refers to a disease in which neurons and nerve fibers are damaged when the spinal cord or its surrounding tissues are subjected to external forces, leading to nerve conduction and motor dysfunction. The incidence and disability rate of spinal cord injury are high. In addition to causing loss of limb movement, sensation and autonomic nervous system function, spinal cord injury can also seriously affect the patient's physical and mental health and quality of life.

[0004] The pathogenesis of spinal cord injury is complex, and one of the key factors is mitochondrial damage. Studies have shown that mitochondrial damage can lead to insufficient neuronal energy supply and excessive ROS production, thereby inducing oxidative stress and causing neuroinflammation. Mitochondria, due to their own double-layer membrane structure, limit the entry of drugs into mitochondria to exert their effects. Therefore, using carriers to deliver drugs into mitochondria and protect damaged mitochondria is expected to be an effective treatment option.

[0005] Hydrogel scaffolds are considered to be a promising drug delivery system with high plasticity, biocompatibility and biodegradability. Studies have shown that implanting hydrogels in the transected area can provide spatial signals to regulate the reorganization of nerve cells. It plays an important role in supporting the severed spinal cord, promoting the reconnection of new neurons and achieving controlled release. Therefore, hydrogel scaffolds can be used as drug carriers to act on the site of spinal cord injury. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a hydrogel scaffold material for spinal cord injury repair and its preparation method and application. In the present invention, liposome-encapsulated AP39 (AP39@LIP) is used as a drug and combined with a GelMA hydrogel scaffold to prepare a hydrogel scaffold material AP39@LIP-GelMA for spinal cord injury repair, which provides a scaffold for the damaged spinal cord, thereby guiding the regeneration of axons in the damaged area, and at the same time, targets the mitochondria in the damaged area to release H2S, alleviate neuronal damage, and thus promote nerve regeneration and functional recovery after spinal cord injury.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a hydrogel scaffold material for repairing spinal cord injury, the hydrogel scaffold material at least comprising:

[0009] A hydrogel scaffold, and a drug loaded on the hydrogel scaffold;

[0010] Wherein, the hydrogel scaffold is a photocurable methacrylated gelatin GelMA hydrogel, which is formed into an individualized scaffold by 3D printing and implanted in situ in the spinal cord injury defect;

[0011] The drug is AP39 encapsulated by liposomes.

[0012] AP39 is a triphenylphosphine-derivatized anethole dithiothione and a mitochondrial-targeted hydrogen sulfide (H2S) donor.

[0013] The second aspect of the present invention provides a method for preparing the hydrogel scaffold material for repairing spinal cord injury described in the first aspect, comprising:

[0014] (1) dissolving a lipid substance in an organic solvent, adding AP39, removing the organic solvent, and forming a film; adding a buffer solution to the film, and hydrating it to form liposome-encapsulated AP39;

[0015] (2) using methacrylic anhydride to react with gelatin to prepare methacrylic acid-acylated gelatin GelMA;

[0016] (3) The liposome-encapsulated AP39 is mixed with methacrylated gelatin GelMA and a photoinitiator, and photocured 3D printing is performed under ultraviolet light to obtain the hydrogel scaffold material for spinal cord injury repair.

[0017] In one or more embodiments, the lipid substance includes soybean lecithin and cholesterol;

[0018] Preferably, the mass ratio of soybean lecithin to cholesterol is (3.5-4.5):1, preferably 4:1.

[0019] In one or more embodiments, in step (1), the organic solvent is selected from ethanol;

[0020] Preferably, the concentration of soybean lecithin in ethanol is 2.3-3 g / L, preferably 2.67 g / L.

[0021] In one or more embodiments, in step (1), the buffer solvent is selected from phosphate buffer.

[0022] The third aspect of the present invention provides the use of the hydrogel scaffold material for spinal cord injury repair described in the first aspect in the preparation of a product for in situ treatment of spinal cord injury.

[0023] In one or more embodiments, the in situ treatment product for spinal cord injury is to implant the hydrogel scaffold material for spinal cord injury repair in situ into the spinal cord injury defect.

[0024] In one or more embodiments, the in situ treatment of spinal cord injury product has at least the following uses:

[0025] a) Enhance neuronal axon regeneration;

[0026] b) Alleviate cell apoptosis caused by spinal cord injury;

[0027] c) Reduce the level of mitochondrial reactive oxygen species (ROS) in the spinal cord after spinal cord injury;

[0028] d) Upregulate the expression of mitochondrial outer membrane protein TOMM20;

[0029] e) Improve motor function recovery after spinal cord injury.

[0030] The beneficial effects of the present invention are:

[0031] (1) The present invention relates to the field of biomaterial technology, and specifically to a hydrogel scaffold material for repairing spinal cord injury, and a preparation method and application thereof. The hydrogel scaffold material for repairing spinal cord injury provided by the present invention can effectively promote the recovery of motor function after spinal cord injury in rats, significantly promote axon regeneration at the injured site, alleviate cell apoptosis caused by spinal cord injury, and upregulate the expression of mitochondrial related proteins. In addition, in in vitro experiments, the ROS level of PC12 cell mitochondria under oxygen glucose deprivation (OGD) increased, while the ATP content decreased. AP39 can reverse the increased ROS level and decreased ATP content of PC12 cell mitochondria induced by OGD. Therefore, the hydrogel scaffold material AP39@LIP-GelMA provided by the present invention can be used as a scaffold at the injured part of the spinal cord, and can also fight against oxidative stress and cell apoptosis caused by spinal cord injury, thereby promoting the repair of the injured site and restoring the motor function of rats after spinal cord injury.

[0032] (2) The present invention prepares AP39 encapsulated in liposomes, i.e., AP39@LIP, by thin film hydration method, and characterizes and tests the AP39@LIP, which shows that the AP39 has the general characteristics of liposomes and can achieve slow release of drugs. Liposome encapsulation can prevent AP39 from being cleared or degraded before it takes effect in the body environment, so liposomes can be used as carriers of AP39.

[0033] (3) Hydrogen sulfide (H2S) is a small molecule endogenous gaseous transmitter that can protect cells from oxidative stress by removing ROS in the mammalian central nervous system. AP39 is a mitochondrial-targeted H2S donor that can release H2S to mitochondria, thereby protecting cells from oxidative stress damage and maintaining the integrity of mitochondrial DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 The characterization results are as follows: AP39@LIP prepared in Example 1, blank liposomes Liposomes prepared in Comparative Example 1, hydrogel scaffold material AP39@LIP-GelMA prepared in Example 2, and hydrogel scaffold material LIP-GelMA prepared in Comparative Example 2; wherein A is a transmission electron micrograph of blank liposomes Liposomes and AP39@LIP; B is the morphology of hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA; C is a scanning electron micrograph of the cross-section of hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA after freeze-drying; and D is the swelling curve and degradation curve of hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA;

[0036] Figure 2 The hydrogel scaffold material AP39@LIP-GelMA can promote the improvement of motor function after spinal cord injury in rats. A is the pie-shaped distribution of BBB scores of rats in each group 28 days after SCI; B is the line graph of BBB scores of rats 1, 3, 5, 7, 14, 21, and 28 days after SCI; C is the analysis of rat footprints on the 14th and 28th days after SCI; D is the statistical analysis of rat footprints 28 days after SCI.

[0037] Figure 3 Verification of the results of hydrogel scaffold material AP39@LIP-GelMA promoting neural regeneration after spinal cord injury in rats; A is immunofluorescence staining to detect spinal cord tissues of rats in each group 28 days after SCI, NF: red, GFAP: green, DAPI: blue; B is statistical analysis of mean fluorescence intensity (MFI) of rat spinal cord immunofluorescence staining; C is immunofluorescence staining to detect spinal cord tissues of rats in each group 28 days after SCI, GAP43: red, GFAP: green, DAPI: blue; D is statistical analysis of MFI of rat spinal cord immunofluorescence staining;

[0038] Figure 4 The hydrogel scaffold material AP39@LIP-GelMA has good biocompatibility results verification; A is TUNEL staining to detect apoptosis of spinal cord tissue cells 28 days after SCI in rats, TUNEL: green, DAPI: blue; B is the statistical analysis of TUNEL+ cells; C is the detection of live / dead cells of neural cells; D is the use of CCK-8 to detect the effects of two hydrogel scaffolds on the cell viability of neurons, microglia or astrocytes;

[0039] Figure 5 The results verify that the AP39@LIP-GelMA hydrogel scaffold reduces the level of mitochondrial ROS in the injured spinal cord of rats after SCI and alleviates the mitochondrial damage caused by SCI; A is the immunohistochemical staining to detect the expression of SOD2 in the spinal cord tissue of each group of rats 28 days after SCI; B is the Western blot detection of the expression of SOD2 and mitochondrial outer membrane protein TOMM20 (Translocase of outer mitochondrial membrane 20) in the spinal cord tissue of each group of rats 28 days after SCI; C is the statistical results of Western blot; D is the use of MitoSOX Red fluorescent probe to detect the level of mitochondrial ROS in PC12 cells, red is MitoSOX Red; blue is DAPI; E is the fluorescence quantitative analysis of mitochondrial ROS in PC12 cells using MitoSOX Red fluorescent probe; F is the determination of ATP content in PC12 cells. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0041] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0043] Example 1

[0044] Preparation of liposome-encapsulated AP39 (AP39@LIP):

[0045] Weigh 40 mg soybean lecithin and 10 mg cholesterol, ultrasonically disperse them in 15 mL ethanol, then add 1 mL AP39 (0.5 mg / mL), stir for 5 min, and use a rotary evaporator to perform rotary evaporation (30°C, 100 rpm) until the ethanol is evaporated to dryness and a liposome film is formed at the bottom of the round-bottom flask. Add 0.01 M phosphate buffered saline (PBS), rotate on a rotary evaporator at room temperature to hydrate it, and obtain AP39@LIP. Take out the liposome solution and store it in a 4°C refrigerator.

[0046] Comparative Example 1

[0047] Weigh 40 mg soybean lecithin and 10 mg cholesterol, ultrasonically disperse them in 15 mL ethanol, and use a rotary evaporator to perform rotary evaporation (30°C, 100 rpm) until the ethanol is evaporated to dryness and a liposome film is formed at the bottom of the round-bottom flask. Add 0.01 M phosphate buffer, rotate on a rotary evaporator at room temperature to hydrate it, obtain blank liposomes, take out the liposome solution, and store it in a 4°C refrigerator.

[0048] Example 2

[0049] First, 0.05g of photoinitiator LAP (2,4,6-trimethylbenzoyl) was dissolved in 20mL of pure water to obtain LAP solution. Then, 1g of GelMA was dissolved in every 20mL of LAP solution to prepare a 5% GelMA hydrogel. Subsequently, the 5% GelMA hydrogel was mixed with liposome-encapsulated AP39 in a ratio of 10:1 and injected into a light-cured biological 3D printer. The curing model was set to a cube with a side length of 1mm, the curing light wavelength was 405nm, and the curing time was set to 10 to 30 seconds. The hydrogel scaffold material AP39@LIP-GelMA for spinal cord injury repair was obtained.

[0050] Comparative Example 2

[0051] First, 0.05g of photoinitiator LAP (2,4,6-trimethylbenzoyl) was dissolved in 20mL of pure water to obtain LAP solution. Then, 1g of GelMA was dissolved in every 20mL of LAP solution to prepare a 5% GelMA hydrogel. Subsequently, the 5% GelMA hydrogel was mixed with blank liposomes in a ratio of 10:1 and injected into a light-cured biological 3D printer. The curing model was set to a cube with a side length of 1mm, the curing light wavelength was 405nm, and the curing time was set to 10-30 seconds. The hydrogel scaffold material LIP-GelMA was obtained.

[0052] Example 3

[0053] The AP39@LIP prepared in Example 1, the blank liposomes Liposomes prepared in Comparative Example 1, the hydrogel scaffold material AP39@LIP-GelMA prepared in Example 2, and the hydrogel scaffold material LIP-GelMA prepared in Comparative Example 2 were characterized. Figure 1 shown.

[0054] Figure 1 A in the middle is a transmission electron micrograph of blank liposomes and AP39@LIP. It can be seen that the two liposomes are hollow vesicle-like and evenly distributed, which indicates that the inclusion of AP39 has no effect on the morphology of the liposomes. Scale bar = 50 nm. Figure 1 B in the middle shows the morphology of the hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA. There is no obvious difference in the appearance of the two 3D printed hydrogel scaffolds. The scale below is 1 mm per grid. Figure 1 Middle C is a scanning electron micrograph of the freeze-dried cross-section of the hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA. The particles in the red dotted circle on the right are blank liposomes and AP39@LIP. The scale bar on the left is 100 μm, and the scale bar on the right is 1 μm. Both hydrogel scaffolds have pores with a diameter of about 50 μm. The presence of these pores enhances the transport of nutrients / metabolic waste and provides space for axon regeneration. Figure 1 Figure D shows the swelling and degradation curves of the hydrogel scaffold materials LIP-GelMA and AP39@LIP-GelMA. The two hydrogel scaffolds swelled to their maximum in about 3 hours under in vitro conditions, indicating that the hydrogel scaffolds quickly absorbed liquid at the damaged site, and the swollen hydrogel scaffolds could better fit with the surrounding tissues to promote tissue repair. The two hydrogel scaffolds could persist for 28 days, indicating that the hydrogel scaffolds could provide structural support in vivo for a long enough time while maintaining stable drug release during treatment.

[0055] Experimental Example 1

[0056] 1. Experimental process:

[0057] (1) Establishment of SCI model: After anesthesia with intraperitoneal injection of 300 mg / kg tribromoethanol, the back of the rat was shaved and disinfected, the skin, fascia and muscle were separated in turn to expose the lamina of the T9-T11 thoracic vertebrae, and then T10 laminectomy was performed. Subsequently, a 1 mm long spinal cord was removed from the right side of the T10 vertebral spinal cord using microscissors. After aspirating the tissue fluid, the pre-prepared hydrogel scaffold material AP39@LIP-GelMA (or hydrogel scaffold material LIP-GelMA) was implanted into the wound. Then gelatin sponge was used to stop bleeding, and then the wound was sutured and disinfected. Paralysis of the right hind limb of the rat was observed after the operation, indicating that the operation was successful. After the operation, ampicillin was injected intraperitoneally every day, and artificial bladder massage was performed to assist urination until spontaneous urination was restored.

[0058] (2) Experimental groups: Adult male SD rats were randomly divided into the following three groups: SCI group, SCI+LIP-GelMA group, and SCI+AP39@LIP-GelMA group.

[0059] (3) BBB score: The BBB score evaluates the recovery of motor function of rats after SCI. The BBB score is used to evaluate the motor activity of rats. Rats are placed in an open space of 125 cm × 125 cm and observed for 4 minutes. The BBB score is recorded on days 1, 3, 5, 7, 14, 21, and 28 after surgery. All evaluations are performed at the same time every day to avoid the influence of circadian rhythm on the experimental results. The BBB score is evaluated by a double-blind method.

[0060] (4) Immunofluorescence staining: Rats were sacrificed 28 days after SCI, and immunofluorescence staining was performed using neurofilament (NF) and growth-associated protein 43 (GAP43) to specifically mark nerve fibers and growing axons in the spinal cord.

[0061] (5) TUNEL staining: Rats were sacrificed 28 days after SCI and apoptotic cells were labeled using the TUNEL (TdT-mediated dUTP Nick-End Labeling) apoptosis detection kit. Apoptotic cells were counted using Image J software. Cell apoptosis rate (%) = number of apoptotic cells / total number of cells × 100%.

[0062] (6) Western blot analysis: Spinal cord tissue was ground in RIPA buffer containing protease / phosphatase inhibitors and PMSF for 15 min, and then centrifuged at 12,000 rpm for 10 min at 4°C using a low-temperature high-speed centrifuge. The supernatant was then taken and the protein concentration was determined using a BCA protein assay kit. Finally, the loading buffer was added and boiled in a metal bath for 10 min and then cooled. The chemiluminescent signal was generated using an ECL kit and then detected using a Tanon imaging system.

[0063] (7) Immunohistochemical staining: Rats were sacrificed 28 days after SCI, and spinal cord tissues were obtained for immunohistochemical staining of superoxide dismutase 2 (SOD2).

[0064] (8) ATP content detection: PC12 cells were cultured in 6-well plates and treated with OGD. They were divided into the Control group, OGD group, OGD+50μM AP39 group, OGD+100μM AP39 group, and OGD+200μM AP39 group. The Control group was not treated with OGD. Cells were collected 6 hours after OGD, and samples were prepared using an ATP detection kit, a standard curve was prepared, and the detection was performed using an ELISA instrument.

[0065] (9) Live / dead cell staining: The live / dead cell staining experiment used a calcein AM / PI double staining kit. The two hydrogel scaffolds were placed in a six-well plate containing PC12 cells and incubated for 30 minutes. The cell suspension was obtained by centrifugation, and the staining solution was added to the cell suspension and incubated for 30 minutes. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were detected under a fluorescence microscope. The effects of the two hydrogel scaffolds on PC12 cells were evaluated after 24 hours and 48 hours, respectively.

[0066] (10) MitoSOX Red detection: PC12 cells were cultured on cell slides, and the fluorescent probe MitoSOX Red was added and incubated for 30 min (37°C). After rinsing with PBS, the slides were sealed with an anti-fluorescence quencher containing DAPI. The staining results were observed under a fluorescence microscope.

[0067] 2. Experimental Results

[0068] (1) Figure 2 The results verify that the hydrogel scaffold material AP39@LIP-GelMA can promote the improvement of motor function after spinal cord injury in rats.

[0069] Figure 2Middle A shows the pie-shaped distribution of BBB scores of rats in each group 28 days after SCI. At 28 days after injury, all rats in the SCI+AP39@LIP-GelMA group scored more than 7 points, and 8 rats scored between 14 and 18 points. N = 12 / group. Figure 2 Middle B is a line graph of BBB scores of rats at 1, 3, 5, 7, 14, 21, and 28 days after SCI. Repeated two-way ANOVA was used to test the differences between the SCI group, SCI+LIP-GelMA group, and SCI+AP39@LIP-GelMA group after injury. Above the line in Figure B, *: Left: SCI+LIP-GelMA vs SCI, Middle: SCI+AP39@LIP-GelMA vs SCI, Right: SCI+AP39@LIP-GelMA vs SCI+LIP-GelMA. The results showed that the functional recovery of rats in the SCI+AP39@LIP-GelMA group after SCI was better than that in the other two groups. N=12 / group. Figure 2 Middle C shows rat footprint analysis on the 14th and 28th days after SCI. Red indicates forelimbs and blue indicates hindlimbs. The results showed that on the 28th day after SCI, the footprints of rats in the SCI+AP39@LIP-GelMA group were significantly reduced. This indicates that the motor function of rats in the SCI+AP39@LIP-GelMA group was restored, N=6 / group. Figure 2 D is the statistical analysis of rat footprints 28 days after SCI, and the results show that the motor function of rats in the SCI+AP39@LIP-GelMA group was significantly restored. Data are expressed as mean ± standard deviation, analyzed by one-way ANOVA and corrected by Bonferroni post hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0070] (2) Figure 3 Verification of the results of the hydrogel scaffold material AP39@LIP-GelMA in promoting nerve regeneration after spinal cord injury in rats.

[0071] Figure 3 A in the middle is immunofluorescence staining to detect the spinal cord tissue of rats in each group 28 days after SCI, NF: red, GFAP: green, DAPI: blue. Scale bar in the left picture = 500 μm, scale bar in the right picture = 50 μm. Figure 3 Middle B is the statistical analysis of the mean fluorescence intensity (MFI) of rat spinal cord immunofluorescence staining. The results show that the MFI of spinal cord NF in the SCI+AP39@LIP-GelMA group was significantly higher than that in the other two groups. This indicates that the hydrogel scaffold material AP39@LIP-GelMA can significantly promote nerve regeneration after SCI in rats, N = 6 / group. Figure 3 Middle C is immunofluorescence staining to detect the spinal cord tissue of rats in each group 28 days after SCI, GAP43: red, GFAP: green, DAPI: blue. Scale bar on the left = 500 μm, scale bar on the right = 50 μm. Figure 3 D in the middle is the statistical analysis of MFI of immunofluorescence staining of rat spinal cord. The MFI of GAP43 in the rat spinal cord of SCI+AP39@LIP-GelMA group was significantly higher than that of the other two groups. This indicates that AP39@LIP-GelMA hydrogel scaffold effectively promotes axon regeneration, N=6 / group. Data are expressed as mean ± standard deviation, analyzed by one-way ANOVA and corrected by Bonferroni post hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0072] (3) Figure 4 The results verified that the hydrogel scaffold material AP39@LIP-GelMA has good biocompatibility.

[0073] Figure 4 A in the middle shows TUNEL staining to detect apoptosis in the spinal cord tissue of rats 28 days after SCI. TUNEL: green, DAPI: blue. Scale bar in the left image = 500 μm, scale bar in the right image = 50 μm. Figure 4 Figure B shows the statistical analysis of TUNEL+ cells, which showed that the TUNEL+ cell rate in the SCI+AP39@LIP-GelMA group was significantly reduced compared with the other two groups, indicating that the AP39@LIP-GelMA hydrogel scaffold reduced the cell apoptosis rate after SCI, N = 6 / group. Figure 4 Middle C is a neuronal live / dead cell assay, and it was found that the addition of these two hydrogel scaffolds had no significant effect on the survival of PC12 cells within 24h and 48h. Live cells: green, dead cells: red. Scale bar = 200μm. Figure 4 D in the middle shows the effect of two hydrogel scaffolds on the cell viability of neurons, microglia or astrocytes detected by CCK-8. The results show that neither hydrogel scaffold causes cytotoxic damage to neurons, microglia or astrocytes. These results indicate that the hydrogel scaffold material AP39@LIP-GelMA has good biocompatibility. The data are expressed as mean ± standard deviation, analyzed by one-way ANOVA and corrected by Bonferroni post hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0074] (4) Figure 5The results verify that the AP39@LIP-GelMA hydrogel scaffold reduces the level of mitochondrial ROS in the injured spinal cord after SCI in rats and alleviates the mitochondrial damage caused by SCI.

[0075] Figure 5 A in the middle is the immunohistochemical staining to detect the expression of SOD2 in the spinal cord tissue of rats in each group 28 days after SCI. The results show that SOD2 is highly expressed in the spinal cord tissue of rats in the AP39@LIP-GelMA group, indicating that the AP39@LIP-GelMA hydrogel scaffold improves the antioxidant capacity of the injured part of the rat. Scale bar on the left = 500μm, scale bar on the right = 50μm. Figure 5 Middle B shows the expression of SOD2 and mitochondrial outer membrane protein TOMM20 (Translocase of outer mitochondrial membrane 20) in the spinal cord tissue of rats in each group 28 days after SCI detected by Western blot. Figure 5 Figure C shows the results of Western blot, which were quantified using ImageJ software, N = 3 / group. The results showed that the expression of SOD2 and TOMM20 in the spinal cord of rats in the AP39@LIP-GelMA group was upregulated. This indicates that the AP39@LIP-GelMA hydrogel scaffold promoted the recovery of mitochondria in the damaged part of the spinal cord of rats after SCI. Figure 5 D in the middle shows the detection of mitochondrial ROS levels in PC12 cells using MitoSOX Red fluorescent probe, red is MitoSOX Red, and blue is DAPI. Scale bar = 20 μm. Figure 5 Middle E is a fluorescence quantitative analysis of mitochondrial ROS in PC12 cells using MitoSOX Red fluorescent probe. The results showed that AP39 treatment significantly reduced the level of mitochondrial ROS in PC12 cells induced by OGD, N=6 / group. Figure 5 F is the determination of ATP content in PC12 cells. The results showed that AP39 could increase ATP content in OGD-treated PC12 cells, N = 3 / group. These results indicate that administration of AP39 can alleviate OGD-induced neuronal oxidative stress and reduce mitochondrial damage. Data are expressed as mean ± standard deviation, analyzed by one-way ANOVA and corrected by Bonferroni post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogel scaffold material for repairing spinal cord injury, characterized in that: The hydrogel scaffold material at least comprises: A hydrogel scaffold, and a drug loaded on the hydrogel scaffold; Wherein, the hydrogel scaffold is a photocurable methacrylated gelatin GelMA hydrogel, which is formed into an individualized scaffold by 3D printing and implanted in situ in the spinal cord injury defect; The drug is AP39 encapsulated by liposomes.

2. The method for preparing the hydrogel scaffold material for repairing spinal cord injury according to claim 1, characterized in that: include: (1) dissolving a lipid substance in an organic solvent, adding AP39, removing the organic solvent, and forming a film; adding a buffer solution to the film, and hydrating it to form liposome-encapsulated AP39; (2) using methacrylic anhydride to react with gelatin to prepare methacrylic acid-acylated gelatin GelMA; (3) The liposome-encapsulated AP39 is mixed with methacrylated gelatin GelMA and a photoinitiator, and photocured 3D printing is performed under ultraviolet light to obtain the hydrogel scaffold material for spinal cord injury repair.

3. The preparation method according to claim 2, characterized in that: In step (1), the lipid substances include soybean lecithin and cholesterol.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the soybean lecithin to cholesterol is (3.5-4.5):1, preferably 4:

1.

5. The preparation method according to claim 3, characterized in that: In step (1), the organic solvent is selected from ethanol.

6. The preparation method according to claim 5, characterized in that: The concentration of the soybean lecithin in ethanol is 2.3-3 g / L, preferably 2.67 g / L.

7. The preparation method according to claim 3, characterized in that: In step (1), the buffer solvent is selected from phosphate buffer.

8. Use of the hydrogel scaffold material for spinal cord injury repair according to claim 1 in the preparation of a product for in situ treatment of spinal cord injury.

9. The use according to claim 8, characterized in that The in situ treatment product for spinal cord injury is to implant the hydrogel scaffold material for spinal cord injury repair in situ into the spinal cord injury defect.

10. The use according to claim 8, characterized in that The in situ treatment product for spinal cord injury has at least the following uses: a) Enhance neuronal axon regeneration; b) Alleviate cell apoptosis caused by spinal cord injury; c) Reduce the level of mitochondrial reactive oxygen species (ROS) in the spinal cord after spinal cord injury; d) Upregulate the expression of mitochondrial outer membrane protein TOMM20; e) Improve motor function recovery after spinal cord injury.

Images