Programmed controllable slow-release bionic spinal cord graft as well as preparation method and application thereof

Abstract

The invention discloses a programmed controllable slow-release bionic spinal cord graft and a preparation method and application thereof, and belongs to the field of medical biomaterials.The preparation method comprises the steps that a micro-nano oriented fiber scaffold with a groove is synthesized on the basis of recombinant NT3, silk fibroin, bovine collagen and a neurotrophic factor NGF; the programmed controllable slow-release bionic spinal cord graft is jointly constructed by combining an acellular matrix and an exosome derived from human umbilical cord mesenchymal stem cells with a programmed slow-release system of GelMA drug-loaded microspheres containing cyclosporine a; multiple technologies such as bionic tissue engineering, a micro-fluidic technology, a drug-loaded microsphere controllable slow-release technology, an acellular matrix and exosome are combined, spinal cord injury repair is facilitated, good biocompatibility is achieved, nerve axons are promoted to grow in an oriented mode, immune regulation can be given in the early stage of spinal cord injury, the nerve regeneration microenvironment after spinal cord injury is improved, and the nerve regeneration effect is improved. The method is beneficial to nerve regeneration and functional reconstruction after spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical biomaterials, and particularly relates to a programmed controlled-release bionic spinal cord graft and its preparation method and application. Background Art

[0002] Spinal cord injury is a severely disabling disease that usually causes irreversible damage to sensory and motor functions. Spinal cord injury can cause a series of complications such as urinary dysfunction, weakness and atrophy of leg muscles, etc., which seriously affect the quality of life of patients.

[0003] After spinal cord injury, neurotransmitter disorders, activation of pro-inflammatory responses, apoptosis, release of neurotoxic saturated lipids, blocked angiogenesis, blocked axon extension, reconstruction of the extracellular matrix structure, and fibrosis and scar formation are the main reasons for the difficulty in spinal cord injury repair. With the development of technologies such as bionic tissue materials, stem cells, and exosomes, in addition, technologies for controlling physical parameters such as electricity, magnetism, and ultrasound are constantly innovated and applied in the field of nerve regeneration. These advanced new strategies and technologies have brought new treatment options for spinal cord injury repair. At present, technologies for treating spinal cord injury using stem cell therapy, biomaterial transplantation, and electromagnetic stimulation have entered the clinical trial stage, but the clinical recovery effect is limited and clinical promotion is very difficult. Given the complex pathological mechanisms after spinal cord injury, a single treatment method is difficult to achieve an ideal treatment effect after spinal cord injury. Summary of the Invention

[0004] Technical problems to be solved:

[0005] Aiming at the deficiencies of the prior art, this application solves the technical problems such as the limited current clinical recovery effect, the difficulty in clinical promotion, and the difficulty in achieving an ideal treatment effect with a single treatment method after spinal cord injury, and provides a programmed controlled-release bionic spinal cord graft and its preparation method and application, which has the functions of spinal cord injury repair and functional reconstruction and is conducive to nerve regeneration after spinal cord injury.

[0006] Technical solutions:

[0007] To achieve the above object, this application is realized through the following technical solutions:

[0008] A programmed controlled-release bionic spinal cord graft, which is prepared by combining a bionic tissue material made from factors beneficial to spinal cord injury repair, silk fibroin, and bovine collagen, an immune drug, acellular matrix, and exosomes to obtain the programmed controlled-release bionic spinal cord graft; the factors beneficial to spinal cord injury repair are recombinant NT3 and neurotrophic factor β-NGF; the bionic tissue material is a micro-nano oriented fiber scaffold with grooves.

[0009] Further, the preparation steps of the micro-nano oriented fiber scaffold with grooves are as follows:

[0010] Step 1: According to the mass ratio of silk fibroin: bovine collagen = 100 - 500:1, mix them in sterile water to prepare a silk fibroin solution with a concentration of 5wt% - 20wt%.

[0011] Step 2: Add 0.3 - 1.5 mL of β-NGF with an initial concentration of 15 μg / mL and 1 -

[0012] 10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL to 0.7 - 2.5 mL of the silk fibroin solution, and pipette and mix well to prepare a silk fibroin blend solution.

[0013] Step 3: Take 100 μL - 1000 μL of the silk fibroin blend solution to wet and fill the gap of the PDMS membrane mold, and then add

[0014] 1 - 5 mL of the silk fibroin blend solution and pour it into the mold with grooves and level it or let it level naturally.

[0015] Step 4: Place the mold with the silk fibroin blend solution flat in a clean petri dish, and let it dry naturally in the laminar flow hood for 12 - 48 h. After demolding, place it in a 70% - 100% ethanol solution for curing for 12 - 48 h, and then let it dry naturally for 12 - 48 h after curing the fibers, thus obtaining the micro-nano oriented fiber scaffold with grooves.

[0016] Step 5: Before use, take 200 - 1000 μL of PBS solution, add 1 - 500 μL of β-NGF solution with an initial concentration of 100 μg / mL and 1 - 10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL, pipette and mix well, then add it to the prepared micro-nano oriented fiber scaffold with grooves, and soak it overnight at 4°C; then straighten the fiber scaffold into a bundle of 10 - 50 fiber scaffolds for subsequent experiments.

[0017] Further, in the second step, the final concentration of recombinant NT3 is 0.15 - 6 ng / mL, and the final concentration of β-NGF is 7.5 - 150 μg / mL.

[0018] Further, the length of the groove in the mold with grooves is 1 - 10 cm, the cross-sectional shape of the groove is an inverted "T" shape, and the inverted "T" shape consists of two parts: a horizontal flange and a vertical web. The total horizontal length of the flange is 50 - 500 μm, the thickness is 20 - 50 μm, the height of the web is 20 - 250 μm, and the width is always less than the total horizontal length of the flange.

[0019] Further, the immune drug is GelMA microspheres CsA-GelMA containing cyclosporine A, wherein the concentration of GelMA microspheres is 5% to 20%, the concentration of cyclosporine A is 10 mM to 200 mM, the acellular matrix is an acellular matrix derived from human umbilical cord mesenchymal stem cells, and the exosomes are exosomes derived from human umbilical cord mesenchymal stem cells.

[0020] Further, the preparation method of the GelMA microspheres CsA-GelMA containing cyclosporine A includes the following steps:

[0021] Step a: Prepare a GelMA solution;

[0022] Step b: Prepare a uniform mixture of cyclosporine A and GelMA solution: Weigh 12 - 120 mg of CsA under sterile conditions and dissolve it in the 5% - 20% GelMA solution prepared in step a, and ultrasonically disperse the solute evenly at 37 °C to finally obtain a CsA-GelMA mixture with a concentration of 10 - 100 mM.

[0023] Step c: Prepare a single emulsion droplet microfluidic device;

[0024] Step d, Generation of cyclosporine A and GelMA microspheres: Place the syringes filled with the CsA-GelMA mixture and corn oil on the syringe pumps respectively, connect them to the microfluidic chip through PE tubes. The flow rate of the syringe filled with the CsA-GelMA mixture is 1 - 10 mL / h, and the flow rate of the syringe filled with corn oil is 20 - 40 mL / h. After observing stable microsphere production under the microscope, start collecting them into a cell culture dish;

[0025] Step e: Cure the collected microspheres under strong ultraviolet irradiation for 30 s - 300 s, check the curing of the microspheres under the microscope, and measure the diameter of the microspheres to be 50 - 250 μm;

[0026] Step f: Elute the corn oil on the surface of the microspheres with 70% - 100% ethanol and collect the CsA-GelMA microspheres.

[0027] A preparation method of a programmed controlled release bionic spinal cord graft. Under the condition of 4°C, CsA-GelMA microspheres and a micro-nano oriented fiber scaffold with grooves are mixed. CsA-GelMA microspheres self-assemble on the micro-nano oriented fiber scaffold with grooves to form a scaffold-microsphere morphology. The acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM, takes the scaffold-microsphere as the axis and wraps around the scaffold-microsphere. The scaffold-microsphere morphology is externally wrapped with the acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM. Among them, the wrapping of the acellular matrix derived from human umbilical cord mesenchymal stem cells wraps 1-10 layers with the micro-nano oriented scaffold with grooves as the axis, and then freeze-dries to prepare the initial morphology of the programmed controlled release bionic spinal cord graft. The exosomes derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-EXO, are collected by the ion exchange method. The initial morphology of the programmed controlled release bionic spinal cord graft after freeze-drying is immersed in the extracted HUC-MSCs-EXO solution to adsorb the exosomes derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-EXO. The time for adsorbing the exosomes derived from human umbilical cord mesenchymal stem cells is 1h to 12h before use, thus forming the programmed controlled release bionic spinal cord graft.

[0028] This application also discloses the application of the programmed controlled release bionic spinal cord graft described in any one of the above or the programmed controlled release bionic spinal cord graft prepared by the above preparation method in the material for repairing spinal cord injury.

[0029] Furthermore, the micro-nano oriented fiber scaffold with grooves is a biodegradable scaffold. The micro-nano oriented fiber scaffold with grooves incorporates recombinant NT3, silk fibroin, nerve growth factor β-NGF, and bovine collagen, with good biocompatibility. Factors beneficial for spinal cord injury repair are released in a programmed and controlled manner as the scaffold material degrades in the body, providing a suitable microenvironment for nerve regeneration, guiding and providing orientation for nerve axon growth, and having a role in nerve protection and promoting nerve regeneration; the GelMA microspheres containing cyclosporine A can release cyclosporine A in a programmed manner and play an immunomodulatory role in the early stage of spinal cord injury; the scaffold-microsphere morphology is externally wrapped with the acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM, providing a beneficial microenvironment for nerve cell growth, as well as guiding and orienting growth effects.

[0030] Principle explanation: The present invention is based on biomaterials, seed cells, active factors, cell matrix and regeneration microenvironment. It forms a micro-nano oriented fiber scaffold with grooves by recombining NT3, silk fibroin fused with bovine collagen and nerve growth factor NGF, combines with microfluidic technology to program-controlled release the immunosuppressive drug cyclosporine A, and then combines with the acellular matrix and exosome technology derived from human umbilical cord mesenchymal stem cells to design and construct a program-controlled release bionic spinal cord graft. A mouse spinal cord hemideficiency experiment was carried out, and the experiment proved that the program-controlled release bionic spinal cord graft provided by the present invention has the effect of repairing spinal cord injury and functional reconstruction in mice. It provides a new treatment plan and strategy for clinical research and treatment, and brings innovative medical technologies for the repair, regeneration and functional reconstruction of human spinal cord injury.

[0031] Beneficial effects:

[0032] The present application provides a program-controlled release bionic spinal cord graft and its preparation method and application. Compared with the prior art, it has the following beneficial effects:

[0033] 1. The present invention constructs a micro-nano oriented fiber scaffold with grooves by using silk fibroin fused with bovine collagen, which has good biocompatibility and biodegradability, and has the function of axon growth guidance;

[0034] 2. The recombinant NT3 and β-NGF are contained in the micro-nano oriented fiber scaffold with grooves of the present invention, and can be slowly released for a long time along with the degradation of the scaffold material, playing the role of nerve protection and promoting nerve regeneration;

[0035] 3. The GelMA microspheres wrapped with cyclosporine A of the present invention realize the program-controlled release of cyclosporine A, and have good biocompatibility and biodegradability, and play a role in regulating immune response in the early stage of spinal cord injury;

[0036] 4. The acellular matrix derived from human umbilical cord mesenchymal stem cells used in the present invention retains various important components and the main framework of the extracellular matrix, which is beneficial to cell adhesion and axon regeneration;

[0037] 5. The exosomes derived from human umbilical cord mesenchymal stem cells used in the present invention have the functions of immune regulation and promoting nerve regeneration in spinal cord injury;

[0038] 6. The acellular matrix and exosomes derived from human umbilical cord mesenchymal stem cells used in the present invention are convenient for the clinical transformation of the program-controlled release bionic spinal cord graft;

[0039] 7. The present invention combines multiple technologies and treats spinal cord injury from multiple aspects, and realizes spinal cord injury repair and functional reconstruction in a mouse spinal cord hemideficiency model. Description of the drawings

[0040] Figure 1 Morphology diagram of the programmed controlled release bionic spinal cord graft after freeze-drying provided by the present invention;

[0041] Figure 2 Diagram showing that the programmed controlled release bionic spinal cord graft provided by the present invention has good biocompatibility. Among them, a is the morphology diagram of the programmed controlled release bionic spinal cord graft provided by the present invention in the spinal cord 3mm semi-defect mice at 3W, 6W, 9W, and 12W; b is the cytotoxicity experiment (CCK8) of the extract of the programmed controlled release bionic spinal cord graft provided by the present invention after culturing PC12 cells for 24h;

[0042] Figure 3 Diagram for the motor function analysis of the programmed controlled release bionic spinal cord graft provided by the present invention in repairing the 3mm semi-defect of the spinal cord of adult B6 mice. Among them, a is the behavioral photo of the experimental mice; b is the hind limb gait imprint of the experimental mice in the catwalk;

[0043] Figure 4 Diagram for the hind limb function analysis of the programmed controlled release bionic spinal cord graft provided by the present invention in repairing the 3mm semi-defect of the spinal cord of adult B6 mice. Among them, a is the morphology diagram of the hind limb muscles of the experimental mice; b is the statistical chart of the wet weight ratio of the muscles ***p < 0.001, ****p < 0.001, Student’s t-test analysis; c is the laser speckle blood flow imaging; d is the statistical chart of the mechanical pain threshold of the right hind limb of the experimental mice *p < 0.05, **p < 0.01, Student’s t-test analysis;

[0044] Figure 5 Diagram for the bladder function analysis of the programmed controlled release bionic spinal cord graft provided by the present invention in repairing the 3mm semi-defect of the spinal cord of adult B6 mice. Among them, a is the morphology diagram of the bladder of the experimental mice at 12W; b is the statistical chart of the bladder volume of the experimental mice ***p < 0.001, ****p < 0.001, Student’s t-test analysis; c is the HE staining of the bladder of the experimental mice at 12W, and the scale bar is 200um;

[0045] Figure 6 Diagram for the spinal cord immunity and glial scar analysis of the programmed controlled release bionic spinal cord graft provided by the present invention in repairing the 3mm semi-defect of the spinal cord of adult B6 mice. Tissue immunofluorescence staining was performed at 3W, 6W, 9W, and 12W after surgery. Green is the microglia labeled by IBA-1, red is the astrocyte labeled by GFAP, and blue is the nucleus labeled by DAPI. The staining scale bar is 200um;

[0046] Figure 7It is an analysis diagram of spinal cord axon and blood vessel regeneration after a 3-mm semi-defect of the spinal cord repaired by a programmed controlled-release bionic spinal cord graft provided by the present invention. Tissue immunofluorescence staining was performed at 3W, 6W, 9W, and 12W after surgery. Red is for axons labeled with TUJ1, green is for blood vessels labeled with CD31, and blue is for cell nuclei labeled with DAPI. The staining scale bar is 200 um. Detailed implementation manners

[0047] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0048] Example 1:

[0049] A programmed controlled-release bionic spinal cord graft, which is prepared by combining a bionic tissue material made from factors beneficial to spinal cord injury repair with an immune drug to obtain a programmed controlled-release bionic spinal cord graft; the factors beneficial to spinal cord injury repair are recombinant NT3, silk fibroin, nerve growth factor β-NGF, and bovine collagen; the bionic tissue material is a micro-nano oriented scaffold with grooves, and the preparation steps of the micro-nano oriented scaffold with grooves are as follows:

[0050] First step: Cut bovine collagen into powder, add water, weigh with the bottle, place it in a sterilizer at 105°C for 10 min, take it out and cool to room temperature, then dry the surface moisture and weigh again to make up for the water lost during sterilization; weigh silk fibroin, add sterile deionized water, add a rotor and stir for 30 min, then add the sterilized bovine collagen. According to the mass ratio of silk fibroin: bovine collagen = 100 - 500:1, continue to stir for 30 min, and let it stand in a 4°C refrigerator to defoam to obtain a silk fibroin solution with a concentration of 5 wt% - 20 wt%.

[0051] Second step: Add 0.3 - 1.5 mL of β-NGF with an initial concentration of 100 μg / mL and 1 - 10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL to 0.7 - 2.5 mL of the silk fibroin solution, and blow and mix evenly to prepare a silk fibroin blend solution; the final concentration of the recombinant NT3 is 0.6 ng / mL, and the final concentration of β-NGF is 15 μg / mL.

[0052] Third step: Sterilize and dry the PDMS membrane mold and cell scraper, take 400 μL of the silk fibroin blend solution to wet and fill

[0053] The gap of the PDMS membrane mold, and then 1.5 mL of the silk fibroin blend solution is added dropwise and poured onto the mold with grooves and leveled or allowed to level naturally. The length of the groove in the mold with grooves is 1-10 cm, and the cross-sectional shape of the groove is an inverted "T" shape, which consists of two parts: a horizontal flange and a vertical web. The total horizontal length of the flange is 50-500 μm, the thickness is 20-50 μm, the height of the web is 20-250 μm, and the width is always less than the total horizontal length of the flange;

[0054] Step 4: Place the mold with the silk fibroin blend solution flat in a clean petri dish, and let it dry naturally in the laminar flow hood for 24 h. After demolding, place it in a 70%-100% ethanol solution for curing for 24 h. After curing the fibers, suck out the absolute ethanol and let it dry naturally in a non-ventilated environment for

[0055] 24 h. After drying, in the laminar flow hood, use a micro straight forceps to peel the fibers from the mold, put them into a clean petri dish, and store them at -20 °C

[0056] in the refrigerator for later use, and then the micro-nano oriented fiber scaffold with grooves is obtained;

[0057] Step 5: Before use, in the laminar flow hood, take 200-1000 μL of PBS solution, add 1-500 μL of β-NGF solution with an initial concentration of 100 μg / mL and 1-10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL, mix well by pipetting, and then add the prepared micro-nano oriented fiber scaffold with grooves, and soak it overnight at 4 °C; then straighten the fiber scaffold into a bundle of 10-50 fiber scaffolds for subsequent experiments.

[0058] Example 2:

[0059] A preparation method of GelMA microspheres CsA-GelMA containing cyclosporine A, comprising the following steps:

[0060] Step a, prepare a GelMA solution: Weigh 0.05 g-2 g of GelMA solid powder under sterile conditions, dissolve it in 1 mL of sterile 1x

[0061] PBS, heat it at 50 °C to assist dissolution, centrifuge at high speed to remove bubbles, and prepare a clear and bubble-free 5%-20% GelMA solution, maintain at 37 °C to prevent condensation; to endow the solution with photo-polymerization properties, add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate LAP to the GelMA solution at a ratio of 1:10000, mix well, and perform a bacteriostatic filtration operation through a 0.22 μm filter head, and store it in the dark for later use;

[0062] Step b, prepare a uniformly mixed solution of CsA cyclosporine A and GelMA solution: Weigh 12-120 mg of CsA under sterile conditions

[0063] Dissolved in the 5%-20% GelMA solution prepared in step a, the solute was ultrasonically and uniformly dispersed at 37°C, and finally a CsA-GelMA mixture with a concentration of 10 -

[0064] 100 mM was obtained;

[0065] Step c, prepare a single-emulsion droplet microfluidic device: Filter the pharmaceutical-grade corn oil through a 0.22 μm filter head for sterilization and reserve it; Assemble the chip in a laminar flow hood. The inner diameter of the inner phase tube is 120 μm, the inner diameter of the outer phase tube is 580 μm, and the inner diameter of the square tube is 1.05 mm. Connect and construct a microfluidic device;

[0066] Step d, generation of cyclosporine A and GelMA microspheres: The CsA-GelMA mixture and corn oil were respectively filled into sterile 1 mL syringes and 5 mL syringes. The syringes filled with the CsA-GelMA mixture and corn oil were respectively placed on the syringe pumps and connected to the microfluidic chip through PE tubes. The flow rate of the syringe filled with the CsA-GelMA mixture was 1-10 mL / h, and the flow rate of the syringe filled with corn oil was 20-40 mL / h. After observing stable microsphere production under the microscope, start collecting them into a cell culture dish;

[0067] Step e: The collected microspheres were cured under strong ultraviolet irradiation for 180 s. Check the completion of microsphere curing under the microscope and measure the diameter of the microspheres to be 100-250 μm;

[0068] Step f: Wash the corn oil on the surface of the microspheres with absolute ethanol and collect the CsA-GelMA microspheres.

[0069] Example 3:

[0070] Self-assembly of CsA-GelMA microspheres and a micro-nano oriented fiber scaffold with grooves, including the following steps:

[0071] Under the condition of 4°C, complete the self-assembly of microspheres and scaffolds in a laminar flow hood. Every 10-50 fibers are bundled into a scaffold, add CsA-GelMA microspheres, and evenly distribute the microspheres inside and on the surface of the scaffold by gently mixing.

[0072] Example 4

[0073] Encapsulate HUC-MSCs-ECM, the steps are as follows:

[0074] Taking the scaffold-microspheres as the axis, the acellular matrix derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-ECM) is wrapped around the scaffold-microspheres. The morphology of the scaffold-microspheres is externally wrapped with the acellular matrix derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-ECM). The wrapping of the acellular matrix derived from human umbilical cord mesenchymal stem cells is wrapped around the micro-nano oriented scaffold with grooves for 1 to 10 layers, providing necessary pathways for the growth of nerve cells, as well as guiding and orienting growth effects.

[0075] Example 5

[0076] Construct the final programmed controlled-release bionic spinal cord graft, and the steps are as follows:

[0077] Freeze-dry to prepare the initial form of the programmed controlled-release bionic spinal cord graft, as Figure 1 shown, which is the morphology diagram of the initial form of the programmed controlled-release bionic spinal cord graft taken by an optical microscope.

[0078] Collect exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-EXO) by ion exchange method. The initial form of the programmed controlled-release bionic spinal cord graft after freeze-drying is immersed in the extracted HUC-MSCs-EXO solution to adsorb exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-EXO). The time for adsorbing exosomes derived from human umbilical cord mesenchymal stem cells is 1 h to 12 h before use to form a programmed controlled-release bionic spinal cord graft.

[0079] Example 6

[0080] Use the programmed controlled-release bionic spinal cord graft to repair a 3 mm semi-defect in the spinal cord of mice:

[0081] Use the programmed controlled-release bionic spinal cord graft to repair a 3 mm semi-defect in the spinal cord of adult B6 mice, and evaluate the recovery of motor function after spinal cord injury repair through BMS behavioral scoring, catwalk, muscle wet weight ratio, lower limb blood flow Doppler imaging, and mechanical pain.

[0082] Establish a 3 mm semi-defect model on the right side of the spinal cord of adult B6 mice and randomly divide them into two groups. The bionic spinal cord graft group is the group that uses the programmed controlled-release bionic spinal cord graft to repair the spinal cord semi-defect of B6 mice (material); the control group is the group that does not receive any intervention after the spinal cord semi-defect of B6 mice (NC).

[0083] Perform BMS behavioral assessment scoring at 3W, 6W, 9W, and 12W after surgery. As Figure 3As shown in the figure, where a is the image of the BMS score at 12 weeks after surgery provided by the embodiments of the present invention. In the group of mice with the programmed controlled sustained-release bionic spinal cord graft, the right hind limb of the mice walked frequently on the soles of the feet, and the trunk was stable when touching the ground and lifting. In the control group, the hind limbs of the mice dragged and could not walk.

[0084] b is the result of the calkwalk footprint experiment. In the group of mice with the programmed controlled sustained-release bionic spinal cord graft, the footprint of the right hind limb was clear, and the sole pressure distribution was uniform. In the control group, the right hind limb of the mice dragged and could not walk on the sole, and basically no footprints were recorded.

[0085] Detecting the wet weight ratio of the target muscle after surgery is an important indicator for evaluating the reconstruction of motor function. As Figure 4 shown in a, as Figure 4 shown in b, it is a schematic diagram of the wet weight ratio result of the target muscle provided by the embodiments of the present invention. As Figure 4 shown in a, it is the appearance morphology diagram of the target muscle of the two groups and its corresponding healthy-side muscle. As Figure 4 shown in b, it is the analysis of the wet weight ratio of the gastrocnemius muscle of the two groups, ***p < 0.001, ****p < 0.001; it can be seen from the figure that at 12 weeks after surgery, the wet weight ratios of the gastrocnemius muscles of the group with the programmed controlled sustained-release bionic spinal cord graft and the control group were 1.0 ± 0.25 and 0.6 ± 0.25 respectively, and there was a statistical difference between the two groups.

[0086] After spinal cord injury, it will cause unstable blood flow in the hind limbs of mice and weakened blood supply function, which affects the recovery of motor function. Laser speckle blood flow imaging of the blood flow situation of the operated hind limb is an index for evaluating the recovery of motor function. As Figure 4 shown in c, it is the laser speckle blood flow imaging diagram provided by the embodiments of the present invention. In the group with the programmed controlled sustained-release bionic spinal cord graft, the blood flow in the right hind limb on the operated side was full, while that in the control group was significantly insufficient.

[0087] Detecting the mechanical pain after surgery is an important indicator for evaluating the recovery of sensory function. As Figure 4 shown in d, it is the statistical chart of mechanical pain provided by the embodiments of the present invention. The pain thresholds of the group with the programmed controlled sustained-release bionic spinal cord graft and the control group were 0.5 ± 0.25 and 1.9 ± 0.25 respectively, and there was a statistical difference between the two groups (***p < 0.001, ****p < 0.001).

[0088] Example 7

[0089] Using the programmed controlled sustained-release bionic spinal cord graft to promote the recovery of bladder function after 3 mm semi-defect of the spinal cord in adult B6 mice:

[0090] Using the programmed controlled sustained-release bionic spinal cord graft to promote the recovery of bladder function after 3 mm semi-defect of the spinal cord in adult B6 mice, and evaluating the recovery of bladder function through the volume of the bladder and HE staining of the bladder.

[0091] Figure 5 In figure a, it shows the external appearance of the bladder of mice in the programmed controlled sustained-release bionic spinal cord graft group and the control group 12 weeks after surgery;

[0092] Figure 5 In figure b, it is a statistical chart of the bladder volume of the programmed controlled sustained-release bionic spinal cord graft group and the control group 12 weeks after surgery. The statistical chart shows that the bladder volumes of the bionic spinal cord graft group and the control group are 0.1±0.01 and 0.2±0.2 respectively, with statistical differences (***p < 0.001, ****p < 0.001). The specific steps are as follows:

[0093] Anesthetize the mice with a mouse anesthetic (tribromoethanol), expose the bladder, slowly inject normal saline into the bladder of the mice until the urine is drained, and record the volume of the directly injected normal saline when the bladder is filled to the maximum.

[0094] As Figure 5 Shown in figure c, it is the HE staining diagram of the bladder 12 weeks after surgery provided in this embodiment;

[0095] Result analysis shows that the bladder wall of the control group becomes thick and the muscle bundles are disordered, while it is significantly improved in the programmed controlled sustained-release bionic spinal cord graft group.

[0096] Example 8

[0097] Using the programmed controlled sustained-release bionic spinal cord graft to reduce the inflammatory response and glial scar after a 3-mm semi-defect of the spinal cord in adult B6 mice:

[0098] Using the programmed controlled sustained-release bionic spinal cord graft to reduce the inflammatory response and glial scar after a 3-mm semi-defect of the spinal cord in adult B6 mice, as Figure 6 Shown, it is the tissue immunofluorescence diagram of microglia (green), astrocytes (red) and cell nuclei (blue) provided in this embodiment.

[0099] Example 9

[0100] Using the programmed controlled sustained-release bionic spinal cord graft to promote axon and blood vessel regeneration after a 3-mm semi-defect of the spinal cord in adult B6 mice:

[0101] Using the programmed controlled sustained-release bionic spinal cord graft to promote axon and blood vessel regeneration after a 3-mm semi-defect of the spinal cord in adult B6 mice, as Figure 7 Shown, it is the tissue immunofluorescence staining diagram of axons (red), blood vessels (green) and cell nuclei (blue) provided in this embodiment.

[0102] The programmed controlled release bionic spinal cord graft used in the present invention does not contain exogenous toxic substances introduced due to the preparation process, has good biocompatibility and biodegradability, uses a biodegradable scaffold, microfluidic technology, extracellular matrix and exosome technology to form a tubular structure, provides a necessary orienting growth effect for nerve cell growth, and the nerve growth factor, cyclosporine A microspheres, acellular matrix and exosomes used can regulate the microenvironment after spinal cord injury and effectively promote nerve regeneration and functional recovery.

[0103] The selected embodiments in the above materials are for easy understanding rather than limiting the process method. Those in the same technical field can easily modify the process flow or migrate it to other cases without creative changes. If these changes also belong to the same type of claims or the same type of technology of the present invention, then the intention of the present invention also includes these changes.

Claims

1. A programmed and controllable sustained-release bionic spinal cord graft, characterized in that: The programmed controlled release bionic spinal cord graft is prepared from a bionic tissue material made of factors beneficial to spinal cord injury repair, silk fibroin, and bovine collagen, combined with an immune drug, a decellularized matrix, and exosomes to obtain the programmed controlled release bionic spinal cord graft; the factors beneficial to spinal cord injury repair are recombinant NT3 and neurotrophic factor β-NGF; the bionic tissue material is a micro-nano oriented fiber scaffold with grooves.

2. The programmable and controllable sustained-release bionic spinal cord graft according to claim 1, wherein The preparation steps of the micro-nano oriented fiber scaffold with grooves are as follows: The first step: Mix silk fibroin and bovine collagen according to a mass ratio of 100-500:1, and prepare a silk fibroin solution with a concentration of 5wt%-20wt% by mixing them in sterile water. The second step: Add 0.3-1.5 mL of β-NGF with an initial concentration of 15 μg / mL and 1-10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL to 0.7-2.5 mL of the silk fibroin solution, and blow and mix evenly to prepare a silk fibroin blend solution. The third step: Take 100 μL-1000 μL of the silk fibroin blend solution to moisten and fill the gap of the PDMS membrane mold, and then drop 1-5 mL of the silk fibroin blend solution into the mold with grooves and level it or let it level naturally. The fourth step: Lay the mold with the silk fibroin blend solution flat in a clean petri dish, air dry it naturally in a laminar flow hood for 12-48 h, demold it and place it in a 70%-100% ethanol solution for curing for 12-48 h, and then air dry it naturally for 12-48 h after curing the fibers to obtain the micro-nano oriented fiber scaffold with grooves. The fifth step: Before use, take 200 μL-1000 μL of PBS solution, add 1-500 μL of β-NGF solution with an initial concentration of 100 μg / mL and 1-10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL, blow and mix evenly, add it to the prepared micro-nano oriented fiber scaffold with grooves, and soak it overnight at 4°C; then straighten the fiber scaffold into a bundle of 10-50 fiber scaffolds for subsequent experiments.

3. The programmable controlled-release bionic spinal cord graft according to claim 2, wherein: In the second step, the final concentration of recombinant NT3 is 0.15-6 ng / mL, and the final concentration of β-NGF is 7.5-150 μg / mL.

4. The programmable controlled-release bionic spinal cord graft according to claim 2, wherein: In the mold with grooves, the length of the groove is 1-10 cm, the cross-sectional shape of the groove is an inverted "T" shape, and the inverted "T" shape consists of a horizontal flange and a vertical web. The total horizontal length of the flange is 50-500 μm, the thickness is 20-50 μm, and the height of the web is 20-250 μm, and the width is always less than the total horizontal length of the flange.

5. The programmable and controllable sustained-release bionic spinal cord graft according to claim 1, characterized in that: The immune drug is GelMA microspheres containing cyclosporine a, CsA-GelMA, where the concentration of GelMA microspheres is 5%-20%, the concentration of cyclosporine a is 10 mM-200 mM, the decellularized matrix is a decellularized matrix derived from human umbilical cord mesenchymal stem cells, and the exosomes are exosomes derived from human umbilical cord mesenchymal stem cells.

6. The programmed controllable sustained-release bionic spinal cord graft according to claim 5, wherein: The preparation method of GelMA microspheres containing cyclosporine a, CsA-GelMA, includes the following steps: Step a: Prepare a GelMA solution; Step b: Prepare a homogeneous mixture of cyclosporine A and GelMA solution: Weigh 12 - 120 mg of CsA under sterile conditions and dissolve it in the 5% - 20% GelMA solution prepared in step a. Ultrasonically disperse the solute evenly at 37 °C to finally obtain a CsA-GelMA mixture with a concentration of 10 - 100 mM. Step c: Prepare a single emulsion droplet microfluidic device; Step d, Generation of cyclosporine A and GelMA microspheres: Place the syringes filled with the CsA-GelMA mixture and corn oil on the syringe pumps respectively, connect them to the microfluidic chip through PE tubes. The flow rate of the syringe filled with the CsA-GelMA mixture is 1 - 10 mL / h, and the flow rate of the syringe filled with corn oil is 20 - 40 mL / h. After observing stable microsphere production under the microscope, start collecting them into a cell culture dish; Step e: The collected microspheres are cured under strong ultraviolet irradiation for 30 s - 300 s. Check the completion of microsphere curing under the microscope and measure the diameter of the microspheres, which is 50 - 250 μm; Step f: Wash the corn oil on the surface of the microspheres with 70% - 100% ethanol and collect the CsA-GelMA microspheres.

7. A method for preparing the programmed controllable sustained-release bionic spinal cord graft according to any one of claims 1-6, characterized in that: Under the condition of 4 °C, the CsA-GelMA microspheres and the micro-nano oriented fiber scaffold with grooves are mixed. The CsA-GelMA microspheres self-assemble on the micro-nano oriented fiber scaffold with grooves to form a scaffold-microsphere morphology. The acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM, takes the scaffold-microsphere as the axis and wraps around the scaffold-microsphere. The scaffold-microsphere morphology is externally wrapped with the acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM. Among them, the wrapping of the acellular matrix derived from human umbilical cord mesenchymal stem cells wraps 1 - 10 layers around the micro-nano oriented scaffold with grooves, and then freeze-dry to prepare the initial morphology of the programmed controlled release bionic spinal cord graft. Use the ion exchange method to collect the exosomes derived from human umbilical cord mesenchymal stem cells, HUC- MSCs-EXO. The initial morphology of the programmed controlled release bionic spinal cord graft after freeze-drying is soaked in the extracted HUC-MSCs-EXO solution to adsorb the exosomes derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-EXO. The time for adsorbing the exosomes derived from human umbilical cord mesenchymal stem cells is 1 h - 12 h before use to form the programmed controlled release bionic spinal cord graft.

8. Use of the programmed controlled release bionic spinal cord graft according to any one of claims 1 - 6 or the programmed controlled release bionic spinal cord graft prepared by the preparation method according to claim 7 in the material for repairing spinal cord injury.

9. The application according to claim 8, wherein: The micro-nano oriented fiber scaffold with grooves is a biodegradable scaffold. The micro-nano oriented fiber scaffold with grooves incorporates recombinant NT3, silk fibroin, nerve growth factor β-NGF, and bovine collagen. It has good biocompatibility, and factors beneficial for spinal cord injury repair are released in a programmed and controllable manner as the scaffold material degrades in the body, providing a suitable microenvironment for nerve regeneration, guiding the growth of nerve axons, and playing a role in nerve protection and promoting nerve regeneration. The GelMA microspheres containing cyclosporine A can release cyclosporine A in a programmed manner, playing an immunomodulatory role in the early stage of spinal cord injury. The scaffold-microsphere morphology is wrapped with acellular matrix derived from human umbilical cord mesenchymal stem cells, HUC-MSCs-ECM, providing a beneficial microenvironment for the growth of nerve cells, as well as guiding and orientation growth effects.

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