Programmed controllable sustained-release bionic spinal cord graft, and preparation method therefor and use thereof

Abstract

The present application belongs to the field of medical biomaterials. Disclosed are a programmed controllable sustained-release bionic spinal cord graft, and a preparation method therefor and the use thereof. The preparation steps include: synthesizing a grooved micro / nano-oriented fiber scaffold based on recombinant NT3, silk fibroin, bovine collagen and neurotrophic factor NGF, and jointly constructing a programmed controllable sustained-release bionic spinal cord graft by combining an acellular matrix and exosome derived from human umbilical cord mesenchymal stem cells with a programmed sustained-release system of a GelMA drug-loaded microsphere containing cyclosporine a. Combined with multiple technologies such as bionic tissue engineering, microfluidic technology, controllable sustained-release technology of drug-loaded microspheres, acellular matrix and exosomes, the present invention is conducive to spinal cord injury repair, has good biocompatibility and promotes the guided growth of nerve axons. The present invention can provide immune regulation at the early stage of spinal cord injury, improve the microenvironment of nerve regeneration after spinal cord injury, and is beneficial to nerve regeneration and functional reconstruction after spinal cord injury.

Description

A programmable, controllable, sustained-release biomimetic spinal cord graft, its preparation method, and its application. Technical Field

[0001] This invention belongs to the field of medical biomaterials technology, specifically relating to a programmed, controllable, sustained-release biomimetic spinal cord graft, its preparation method, and its application. Background Technology

[0002] Spinal cord injury is a severely disabling disease that usually leads to irreversible damage to sensory and motor functions. Spinal cord injury can cause a range of complications, such as urinary dysfunction, leg muscle weakness and atrophy, which significantly impact a patient's quality of life.

[0003] Following spinal cord injury (SLE), neurotransmitter imbalances, activation of pro-inflammatory responses, apoptosis, release of neurotoxic saturated lipids, impaired angiogenesis, hindered axonal elongation, extracellular matrix reconstruction, fibrosis, and scar formation are the main reasons for the difficulty in SLE repair. With the development of biomimetic tissue materials, stem cells, and exosomes, and the continuous innovation and application of technologies controlling physical parameters such as electricity, magnetism, and ultrasound in the field of nerve regeneration, these advanced new strategies and technologies have brought new treatment options for SLE repair. Currently, technologies utilizing stem cell therapy, biomaterial transplantation, and electromagnetic stimulation to treat SLE have entered the clinical trial stage; however, clinical recovery effects are limited, and clinical promotion is difficult. Given the complex pathological mechanisms following SLE, single treatment methods are unlikely to achieve ideal therapeutic effects. Summary of the Invention

[0004] Technical problems to be solved:

[0005] This application addresses the shortcomings of existing technologies, solving technical problems such as limited clinical recovery effects, difficulties in clinical promotion, and the inability of single treatment methods to achieve ideal therapeutic effects after spinal cord injury. It provides a programmed, controllable, sustained-release bionic spinal cord graft, its preparation method, and its application, which has the functions of spinal cord injury repair and functional reconstruction, and is beneficial to nerve regeneration after spinal cord injury.

[0006] Technical solution:

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] A programmed, controllable, sustained-release biomimetic spinal cord graft is provided. The graft is made from biomimetic tissue materials derived from factors beneficial to spinal cord injury repair, silk fibroin, and bovine collagen, combined with immunomodulatory drugs, decellularized matrix, and exocrine agents. The factors beneficial to spinal cord injury repair are recombinant NT3 and neurotrophic factor β-NGF. The biomimetic tissue material is a grooved micro / nano oriented fiber scaffold.

[0009] Furthermore, the fabrication steps of the grooved micro / nano oriented fiber scaffold are as follows:

[0010] Step 1: Prepare a silk fibroin solution with a concentration of 5wt% to 20wt% by mixing the silk fibroin and bovine collagen in a mass ratio of 100 to 500:1 in sterile water.

[0011] Step 2: Add 0.3-1.5 mL of β-NGF (initial concentration 15 μg / mL) and 1- to 0.7-2.5 mL of silk fibroin solution.

[0012] 10 μL of recombinant NT3 with an initial concentration of 0.6 ng / mL was mixed by pipetting to prepare a silk fibroin blend solution;

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

[0014] Pour 1-5 mL of silk fibroin blend solution into a grooved mold and level it or let it flow naturally.

[0015] Step 4: Place the mold containing the silk fibroin blend solution in a clean petri dish and air dry it in a clean bench for 12-48 hours. After demolding, place it in a 70%-100% ethanol solution to cure for 12-48 hours. After curing the fibers, air dry it in a clean bench for 12-48 hours to obtain a 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, mix well by blowing, add the prepared grooved micro / nano oriented fiber scaffold, and soak overnight at 4°C; then straighten the fiber scaffold into bundles of 10-50 fiber scaffolds for subsequent experiments.

[0017] Furthermore, 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] Furthermore, in the mold with the groove, the groove length is 1-10cm, the cross-sectional shape of the groove is an inverted "T" shape, the inverted "T" shape is composed of two parts: a horizontal flange and a vertical web. The total horizontal length of the flange is 50-500μm, the thickness is 20-50μm, the height of the web is 20-250μm, and the width is always less than the total horizontal length of the flange.

[0019] Furthermore, the immunomodulatory 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 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.

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

[0021] Step a: Prepare the GelMA solution;

[0022] Step b: Prepare a homogeneous mixture of cyclosporine a and GelMA solution: Under aseptic conditions, weigh 12-120 mg CsA and dissolve it in the 5%-20% GelMA solution prepared in step a. Disperse the solute uniformly by sonication at 37°C to 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, Cyclosporine a and GelMA microsphere generation: The syringes containing CsA-GelMA mixture and corn oil were respectively placed on the injection pump and connected to the microfluidic chip through PE tubing. The flow rate of the syringe containing CsA-GelMA mixture was 1-10 mL / h, and the flow rate of the syringe containing corn oil was 20-40 mL / h. After observing stable microsphere production under a microscope, the microspheres were collected into cell culture dishes.

[0025] Step e: Collect the microspheres and cure them under strong ultraviolet irradiation for 30s-300s. Observe the curing of the microspheres under a microscope and measure the diameter of the microspheres (50-250μm).

[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 method for preparing a programmable, controllable, sustained-release biomimetic spinal cord graft involves mixing CsA-GelMA microspheres and a grooved micro / nano-oriented fiber scaffold at 4°C. CsA-GelMA microspheres self-assemble on the grooved micro / nano-oriented fiber scaffold to form a scaffold-microsphere morphology. Human umbilical cord mesenchymal stem cell-derived decellularized matrix (HUC-MSCs-ECM) is then wrapped around the scaffold-microsphere morphology, with the scaffold-microsphere morphology serving as the axis. The scaffold-microsphere morphology is thus encapsulated by the human umbilical cord mesenchymal stem cell-derived decellularized matrix (HUC-MSCs-ECM). The encapsulation of the human umbilical cord mesenchymal stem cell-derived decellularized matrix is ​​characterized by... A grooved micro-nano oriented scaffold is wrapped with 1-10 layers and freeze-dried to prepare the initial form of a programmable, controllable, sustained-release biomimetic spinal cord graft. Human umbilical cord mesenchymal stem cell-derived exosomes HUC-MSCs-EXO are collected using an ion exchange method. After freeze-drying, the initial form of the programmable, controllable, sustained-release biomimetic spinal cord graft is immersed in the extracted HUC-MSCs-EXO solution to adsorb the human umbilical cord mesenchymal stem cell-derived exosomes HUC-MSCs-EXO. The adsorption time of the human umbilical cord mesenchymal stem cell-derived exosomes is 1-12 hours before use, thus forming a programmable, controllable, sustained-release biomimetic spinal cord graft.

[0028] This application also discloses the application of any of the above-described programmed controllable sustained-release biomimetic spinal cord grafts or programmed controllable sustained-release biomimetic spinal cord grafts prepared by the above-described methods in materials for repairing spinal cord injuries.

[0029] Furthermore, the grooved micro / nano oriented fiber scaffold is a biodegradable scaffold. This grooved micro / nano oriented fiber scaffold integrates recombinant NT3, silk fibroin, neurotrophic factor β-NGF, and bovine collagen, exhibiting good biocompatibility. Factors beneficial for spinal cord injury repair are released in a programmed and controllable manner as the scaffold material degrades in vivo, providing a suitable microenvironment for nerve regeneration, guiding nerve axon growth, providing neuroprotection, and promoting nerve regeneration. The GelMA microspheres containing cyclosporine a can programmatically release cyclosporine a, exerting an immunomodulatory effect in the early stages of spinal cord injury. The scaffold-microsphere morphology is encapsulated with decellularized matrix HUC-MSCs-ECM derived from human umbilical cord mesenchymal stem cells, providing a beneficial microenvironment for nerve cell growth, as well as guiding and oriented growth.

[0030] Explanation of Principle: This invention is based on biomaterials, seed cells, active factors, cell matrix, and regenerative microenvironment. It utilizes recombinant NT3, silk fibroin fused with bovine collagen and neurotrophic factor NGF to form a grooved micro / nano oriented fiber scaffold. Combined with microfluidic technology for programmed sustained release of the immunomodulatory drug cyclosporine a, and further integrated with decellularized matrix derived from human umbilical cord mesenchymal stem cells and exosome technology, a programmed, controllable, sustained-release biomimetic spinal cord graft was designed and constructed. Experiments were conducted on mouse spinal cord hemi-ischemia, demonstrating that the controllable, sustained-release biomimetic spinal cord graft provided by this invention can repair spinal cord injury and restore function in mice. This provides a new treatment plan and strategy for clinical research and treatment, bringing innovative medical technologies to the repair, regeneration, and functional reconstruction of human spinal cord injury. Beneficial effects:

[0031] This application provides a programmable, controllable, sustained-release biomimetic spinal cord graft, its preparation method, and its application. Compared with the prior art, it has the following advantages:

[0032] 1. This invention uses silk fibroin fused with bovine collagen to construct a grooved micro / nano oriented fiber scaffold, which has good biocompatibility and biodegradability, and also has axon growth guiding function;

[0033] 2. The grooved micro / nano oriented fiber scaffold of the present invention contains recombinant NT3 and β-NGF, which can be released slowly over a long period of time as the scaffold material degrades, thereby exerting neuroprotective and nerve regeneration effects;

[0034] 3. The GelMA microspheres encapsulating cyclosporine a used in this invention achieve programmed sustained release of cyclosporine a, and have good biocompatibility and biodegradability, and play a role in regulating the immune response in the early stage of spinal cord injury.

[0035] 4. The decellularized matrix derived from human umbilical cord mesenchymal stem cells used in this invention retains various important components and the main framework of the extracellular matrix, which is conducive to cell adhesion and axon regeneration;

[0036] 5. The exosomes derived from human umbilical cord mesenchymal stem cells used in this invention have immunomodulatory and nerve regeneration-promoting effects in spinal cord injury;

[0037] 6. The decellularized matrix and exosomes derived from human umbilical cord mesenchymal stem cells used in this invention facilitate the clinical translation of programmed, controllable, and sustained-release biomimetic spinal cord transplants;

[0038] 7. This invention employs a combination of multiple technologies to treat spinal cord injury from multiple perspectives, achieving spinal cord injury repair and functional reconstruction in a mouse model of spinal cord hemiparesis. Attached image description:

[0039] Figure 1 is a morphological diagram of the freeze-dried, programmed, controllable, sustained-release biomimetic spinal cord transplant provided by the present invention.

[0040] Figure 2 shows the good biocompatibility of the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention. In Figure 2, a shows the morphology of the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention in mice with a 3mm spinal cord hemisphere defect at 3 weeks, 6 weeks, 9 weeks, and 12 weeks. Figure 2 shows the cytotoxicity experiment (CCK8) of the extract of the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention after culturing PC12 cells for 24 hours.

[0041] Figure 3 is a motor function analysis diagram of repairing a 3mm half-defect of the spinal cord in an adult B6 mouse with a programmed controllable sustained-release bionic spinal cord graft provided by the present invention. In this figure, a is a behavioral photograph of the experimental mouse; b is the gait imprint of the hind limbs of the experimental mouse in a catwalk.

[0042] Figure 4 shows the hindlimb function analysis of the adult B6 mouse with a 3mm hemi-defect spinal cord repaired by the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention. In Figure 4, a is the morphology of the hindlimb muscles of the experimental mouse; b is the statistical analysis of the muscle wet weight ratio (***p<0.001, ****p<0.001, Student's t-test analysis); c is laser speckle blood flow imaging; d is the statistical analysis of the mechanical pain threshold of the right hindlimb of the experimental mouse (*p<0.05, **p<0.01, Student's t-test analysis).

[0043] Figure 5 shows the bladder function analysis of the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention in repairing a 3mm hemi-defect of the spinal cord in adult B6 mice. In the figure, a is the bladder morphology of the 12-week-old experimental mouse; b is the bladder volume statistics of the 12-week-old experimental mouse, p < 0.001, p < 0.001, Student's t-test analysis; c is the HE staining of the bladder of the 12-week-old experimental mouse with a scale bar of 200 μm.

[0044] Figure 6 shows the spinal cord immunological and glial scar analysis of the 3mm half-defect spinal cord in adult B6 mice repaired by the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention. Immunofluorescence staining was performed at 3, 6, 9 and 12 weeks postoperatively. Green represents IBA-1 labeled microglia, red represents GFAP labeled astrocytes, and blue represents DAPI labeled cell nuclei. The staining scale bar is 200 μm.

[0045] Figure 7 is an analysis diagram of spinal cord axon and angiogenesis regeneration in adult B6 mice with a 3mm spinal cord hemisphere defect repaired by the programmed controllable sustained-release biomimetic spinal cord graft provided by the present invention. Immunofluorescence staining of tissues was performed at 3, 6, 9 and 12 weeks after surgery. Red represents TUJ1 labeled axons, green represents CD31 labeled blood vessels, and blue represents DAPI labeled cell nuclei. The staining scale bar is 200um. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0047] Example 1:

[0048] A programmed, controllable, sustained-release biomimetic spinal cord graft is prepared from biomimetic tissue materials derived from factors beneficial to spinal cord injury repair, combined with immunotherapies to achieve the programmed, controllable, sustained-release biomimetic spinal cord graft. The factors beneficial to spinal cord injury repair include recombinant NT3, silk fibroin, neurotrophic factor β-NGF, and bovine collagen. The biomimetic tissue material is a grooved micro / nano-oriented scaffold, and the preparation steps of the grooved micro / nano-oriented scaffold are as follows:

[0049] Step 1: Cut bovine collagen into powder, add water, weigh the mixture in the bottle, place it in a 105℃ sterilizer for 10 minutes, remove it, cool it to room temperature, wipe off the surface moisture, and weigh it again to replenish the water lost during sterilization; weigh silk fibroin, add sterile deionized water, add it to a rotor and stir for 30 minutes, then add the sterilized bovine collagen, and mix silk fibroin:bovine collagen in a mass ratio of 100-500:1, continue stirring for 30 minutes, and let it stand in a 4℃ refrigerator to defoam, obtaining a silk fibroin solution with a concentration of 5wt%-20wt%;

[0050] Step 2: 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 silk fibroin solution, and mix thoroughly by pipetting to prepare a silk fibroin blend solution; the final concentration of recombinant NT3 is 0.6 ng / mL and the final concentration of β-NGF is 15 μg / mL.

[0051] Step 3: Sterilize and dry the PDMS membrane mold and cell scraper. Take 400 μL of silk fibroin blend solution to moisten and fill the gaps of the PDMS membrane mold. Then add 1.5 mL of silk fibroin blend solution and pour it into the grooved mold and level it or let it flow naturally. The groove in the grooved mold has a groove length of 1 to 10 cm and a groove cross-sectional shape of an inverted "T". The inverted "T" shape consists of two parts: a horizontal flange and a vertical web. The total horizontal length of the flange is 50 to 500 μm and the thickness is 20 to 50 μm. The height of the web is 20 to 250 μm and the width is always less than the total horizontal length of the flange.

[0052] Step 4: Place the mold containing the silk fibroin blend solution in a clean petri dish and air dry it in a laminar flow hood for 24 hours. After demolding, place it in a 70%–100% ethanol solution to solidify for 24 hours. After the fiber is solidified, remove the anhydrous ethanol and air dry it in a non-ventilated environment for 24 hours. After drying, in a laminar flow hood, use micro-straight forceps to peel the fiber from the mold and place it in a clean petri dish. Store it in a -20°C freezer for later use. This yields a grooved micro / nano oriented fiber scaffold.

[0053] Step 5: Before use, in a clean bench, 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 blowing, then add the prepared grooved micro / nano oriented fiber scaffold, and soak overnight at 4°C; then straighten the fiber scaffold into bundles of 10-50 fiber scaffolds for subsequent experiments.

[0054] Example 2:

[0055] The preparation method of GelMA microspheres CsA-GelMA containing cyclosporine a includes the following steps:

[0056] Step a, prepare GelMA solution: Under aseptic conditions, weigh 0.05g-2g of GelMA solid powder, dissolve it in 1mL of sterile 1x PBS, heat at 50℃ to aid dissolution, centrifuge at high speed to defoam, and prepare a clear, foam-free 5%-20% GelMA solution. Maintain the temperature at 37℃ to prevent condensation. To impart photopolymerization properties to the solution, add lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) to the GelMA solution at a ratio of 1:10000, mix thoroughly, and filter through a 0.22μm filter head for bacterial filtration. Store in the dark for later use.

[0057] Step b, prepare a homogeneous mixture of CsA cyclosporine a and GelMA solution: under aseptic conditions, weigh 12-120 mg of CsA and dissolve it in the 5%-20% GelMA solution prepared in step a, and solute is uniformly dispersed by ultrasonication at 37°C to finally obtain a CsA-GelMA mixture with a concentration of 10-100 mM.

[0058] Step c, prepare a single emulsion droplet microfluidic device: filter pharmaceutical-grade corn oil through a 0.22μm filter head for bacterial removal and set aside; assemble the chip in a clean bench, with the inner diameter of the inner phase tube being 120μm, the inner diameter of the outer phase tube being 580μm, and the inner diameter of the square tube being 1.05mm, and connect them to form a microfluidic device.

[0059] Step d, Cyclosporine a and GelMA microsphere generation: CsA-GelMA mixture and corn oil were respectively filled into sterile 1mL syringes and 5mL syringes. The syringes containing CsA-GelMA mixture and corn oil were respectively placed on the injection pump and connected to the microfluidic chip through PE tubing. The flow rate of the syringe containing CsA-GelMA mixture was 1-10mL / h, and the flow rate of the syringe containing corn oil was 20-40mL / h. After observing stable microsphere production under a microscope, the microspheres were collected into cell culture dishes.

[0060] Step e: Collect the microspheres and cure them under strong ultraviolet irradiation for 180s. Observe the curing of the microspheres under a microscope and measure the diameter of the microspheres (100-250μm).

[0061] Step f: Wash the corn oil off the surface of the microspheres with anhydrous ethanol and collect the CsA-GelMA microspheres.

[0062] Example 3:

[0063] The self-assembly of CsA-GelMA microspheres with grooved micro / nano oriented fiber scaffolds includes the following steps:

[0064] The self-assembly of microspheres and scaffolds was completed in a clean bench at 4°C. Each scaffold consisted of 10-50 fibers, and CsA-GelMA microspheres were added. The microspheres were then gently mixed to distribute them evenly inside and on the surface of the scaffold.

[0065] Example 4

[0066] The steps for packaging HUC-MSCs-ECM are as follows:

[0067] Human umbilical cord mesenchymal stem cell-derived decellularized matrix HUC-MSCs-ECM is wrapped around a scaffold-microsphere axis. The scaffold-microsphere morphology encapsulates the human umbilical cord mesenchymal stem cell-derived decellularized matrix HUC-MSCs-ECM. The encapsulation of the human umbilical cord mesenchymal stem cell-derived decellularized matrix is ​​wrapped in 1-10 layers with grooved micro-nano oriented scaffold as the axis, providing the necessary pathway for nerve cell growth and playing a guiding and oriented growth role.

[0068] Example 5

[0069] The steps for constructing the final programmable, controllable, sustained-release biomimetic spinal cord graft are as follows:

[0070] Freeze-drying was used to prepare the initial morphology of the programmed controllable sustained-release bionic spinal cord transplant, as shown in Figure 1, which is a morphological image of the initial morphology of the programmed controllable sustained-release bionic spinal cord transplant taken by an optical microscope.

[0071] Human umbilical cord mesenchymal stem cell-derived exosomes HUC-MSCs-EXO were collected using an ion exchange method. After freeze-drying, the initial morphology of the programmed controllable sustained-release biomimetic spinal cord graft was immersed in the extracted HUC-MSCs-EXO solution to adsorb the human umbilical cord mesenchymal stem cell-derived exosomes HUC-MSCs-EXO. The adsorption time of the human umbilical cord mesenchymal stem cell-derived exosomes was 1 hour to 12 hours before use, thus forming a programmed controllable sustained-release biomimetic spinal cord graft.

[0072] Example 6

[0073] Repairing a 3mm hemi-defect of the spinal cord in mice using a programmed, controlled, sustained-release biomimetic spinal cord graft:

[0074] A programmed, controllable, sustained-release biomimetic spinal cord graft was used to repair a 3mm hemi-defect of the spinal cord in adult B6 mice. The recovery of motor function after spinal cord injury repair was assessed using BMS behavioral scores, catwalk, muscle wet weight ratio, lower limb blood flow Doppler imaging, and mechanical pain.

[0075] A 3mm right spinal cord hemi-defect model was established in adult B6 mice, and the mice were randomly divided into two groups. The biomimetic spinal cord graft group (material) was used to repair the spinal cord hemi-defect in B6 mice with a programmed, controllable, sustained-release biomimetic spinal cord graft; the control group (NC) was the B6 mice that received no intervention after spinal cord hemi-defect.

[0076] BMS behavioral assessments were performed at 3, 6, 9, and 12 weeks post-surgery. As shown in Figure 3, where a represents the BMS score image at 12 weeks post-surgery provided in this embodiment of the invention, mice in the programmed controllable sustained-release bionic spinal cord graft group exhibited frequent plantar walking on their right hind limbs, with trunk stability during ground contact and lifting. Control group mice dragged their hind limbs and were unable to walk.

[0077] b represents the results of the Calkwalk footprint experiment. The programmed, controllable, sustained-release bionic spinal cord graft group showed clear footprints on the right hind limb with uniform pressure distribution. The control group mice dragged their right hind limbs and could not walk on their soles, with virtually no footprints recorded.

[0078] Postoperative target muscle wet weight ratio is an important indicator for evaluating motor function reconstruction. Figures 4a and 4b show the target muscle wet weight ratio results provided in this embodiment of the invention. Figure 4a shows the morphological appearance of the two target muscles and their corresponding healthy side muscles. Figure 4b shows the wet weight ratio analysis of the two gastrocnemius muscles (***p<0.001, ****p<0.001). As can be seen from the figures, at 12 weeks post-surgery, the wet weight ratios of the gastrocnemius muscles in the programmed controllable sustained-release bionic spinal cord graft group and the control group were 1.0±0.25 and 0.6±0.25, respectively, showing a statistically significant difference between the two groups.

[0079] Spinal cord injury can lead to unstable blood flow and weakened blood supply in the hind limbs of mice, affecting the recovery of motor function. Laser speckle flow imaging of the blood flow in the operated hind limb is an indicator for assessing the recovery of motor function. As shown in Figure 4c, the laser speckle flow imaging diagram provided in this embodiment of the invention shows that the right hind limb of the operated side is well-filled in the programmed controllable sustained-release bionic spinal cord graft group, while the blood flow in the control group is significantly insufficient.

[0080] Postoperative mechanical pain detection is an important indicator for assessing the recovery of sensory function. As shown in Figure 4d, it is a statistical chart of mechanical pain provided by the embodiment of the present invention. The pain thresholds of the programmed controllable sustained-release bionic spinal cord graft group 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).

[0081] Example 7

[0082] Using programmed, controlled, sustained-release biomimetic spinal cord grafts to promote bladder function recovery in adult B6 mice following a 3mm spinal cord hemisphere defect:

[0083] A programmed, controlled, sustained-release biomimetic spinal cord graft was used to promote bladder function recovery in adult B6 mice with a 3mm spinal cord hemi-defect. Bladder function recovery was assessed by bladder volume and HE staining.

[0084] Figure 5a shows the bladder appearance of mice in the programmed controlled-release bionic spinal cord transplant group and the control group 12 weeks after surgery.

[0085] Figure 5b shows the bladder volume statistics of the programmed controlled-release bionic spinal cord graft group and the control group 12 weeks post-surgery. The statistics show that the bladder volumes of the bionic spinal cord graft group and the control group were 0.1±0.01 and 0.2±0.2, respectively, which were statistically different (***p<0.001, ****p<0.001). The specific steps are as follows:

[0086] Mice were anesthetized with a mouse anesthetic (tribromoethanol), and their bladders were exposed. Physiological saline was slowly injected into the bladders of mice that had urinated, and the volume of physiological saline injected was recorded when the bladder was fully filled.

[0087] As shown in Figure 5c, this is an HE staining image of the bladder 12 weeks post-surgery provided in this embodiment.

[0088] Results analysis showed that the bladder wall thickened and muscle bundles were disordered in the control group, while the programmed controllable sustained-release bionic spinal cord graft group showed significant improvement.

[0089] Example 8

[0090] Using programmed, controlled-release biomimetic spinal cord grafts to reduce inflammation and glial scarring following a 3mm spinal cord hemisphere defect in adult B6 mice:

[0091] The use of a programmed, controllable, sustained-release biomimetic spinal cord graft reduced inflammation and glial scarring in adult B6 mice following a 3mm spinal cord hemisphere defect. Figure 6 shows an immunofluorescence image of microglia (green), astrocytes (red), and cell nuclei (blue) tissues provided in this embodiment.

[0092] Example 9

[0093] Using programmed, controlled, sustained-release biomimetic spinal cord grafts to promote axonal and vascular regeneration in adult B6 mice with a 3mm spinal cord hemisphere defect:

[0094] The use of a programmed, controllable, sustained-release biomimetic spinal cord graft to promote axonal and vascular regeneration in adult B6 mice with a 3mm spinal cord hemisphere defect is shown in Figure 7, which is a tissue immunofluorescence staining image of axons (red), blood vessels (green), and cell nuclei (blue) provided in this embodiment.

[0095] The programmed, controllable, sustained-release biomimetic spinal cord graft used in this invention does not contain any exogenous toxic substances introduced by the manufacturing process, and has good biocompatibility and biodegradability. It uses a biodegradable scaffold, microfluidic technology, extracellular matrix and exosome technology to form a tubular structure, which provides the necessary directional growth for nerve cell growth. The nerve growth factor, cyclosporine a microspheres, decellularized matrix and exosomes used can regulate the microenvironment after spinal cord injury, effectively promoting nerve regeneration and functional recovery.

[0096] The embodiments selected in the above materials are for ease of understanding and not for limiting the process method. Those skilled in the art can easily modify the process flow or transfer it to other cases without inventive change. If these modifications also fall under the category of similar claims or similar technology of this invention, then the intent of this invention also includes these modifications.

Claims

1. A programmable, controllable, sustained-release biomimetic spinal cord graft, characterized in that: The programmed, controllable, sustained-release biomimetic spinal cord graft is made from biomimetic tissue materials derived from factors beneficial to spinal cord injury repair, silk fibroin, and bovine collagen, combined with immunomodulatory drugs, decellularized matrix, and exocrine agents to obtain the programmed, controllable, sustained-release biomimetic spinal cord graft; the factors beneficial to spinal cord injury repair are recombinant NT3 and neurotrophic factor β-NGF; the biomimetic tissue material is a grooved micro / nano oriented fiber scaffold.

2. The programmed, controllable, sustained-release bionic spinal cord graft according to claim 1, characterized in that, The fabrication steps of the grooved micro / nano oriented fiber scaffold are as follows: Step 1: Prepare a silk fibroin solution with a concentration of 5wt% to 20wt% by mixing the silk fibroin and bovine collagen in a mass ratio of 100 to 500:1 in sterile water. Step 2: 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 silk fibroin solution, and mix well by pipetting to prepare a silk fibroin blend solution. Step 3: Take 100μL to 1000μL of silk fibroin blend solution to wet and fill the gaps in the PDMS membrane mold, then add 1 to 5mL of silk fibroin blend solution and pour it into the mold with grooves and spread it out or let it flow naturally. Step 4: Place the mold containing the silk fibroin blend solution in a clean petri dish and air dry it in a clean bench for 12-48 hours. After demolding, place it in a 70%-100% ethanol solution to cure for 12-48 hours. After curing the fibers, air dry it in a clean bench for 12-48 hours to obtain a micro-nano oriented fiber scaffold with grooves. 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, mix well by blowing, add the prepared grooved micro / nano oriented fiber scaffold, and soak overnight at 4°C; then straighten the fiber scaffold into bundles of 10-50 fiber scaffolds for subsequent experiments.

3. The programmed, controllable, sustained-release biomimetic spinal cord graft according to claim 2, characterized in that: 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 programmed, controllable, sustained-release biomimetic spinal cord graft according to claim 2, characterized in that: The groove in the mold has a groove length of 1 to 10 cm and a cross-sectional shape of an inverted "T". The inverted "T" is composed of a horizontal flange and a vertical web. The total horizontal length of the flange is 50 to 500 μm and the thickness is 20 to 50 μm. The height of the web is 20 to 250 μm and the width is always less than the total horizontal length of the flange.

5. The programmed, controllable, sustained-release bionic spinal cord graft according to claim 1, characterized in that: The immunomodulatory 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 decellularized matrix is ​​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, characterized in that: The preparation method of GelMA microspheres CsA-GelMA containing cyclosporine a includes the following steps: Step a: Prepare the GelMA solution; Step b: Prepare a homogeneous mixture of cyclosporine a and GelMA solution: Under aseptic conditions, weigh 12-120 mg CsA and dissolve it in the 5%-20% GelMA solution prepared in step a. Disperse the solute uniformly by sonication at 37°C to obtain a CsA-GelMA mixture with a concentration of 10-100 mM. Step c: Prepare a single emulsion droplet microfluidic device; Step d, Cyclosporine a and GelMA microsphere generation: The syringes containing CsA-GelMA mixture and corn oil were respectively placed on the injection pump and connected to the microfluidic chip through PE tubing. The flow rate of the syringe containing CsA-GelMA mixture was 1-10 mL / h, and the flow rate of the syringe containing corn oil was 20-40 mL / h. After observing stable microsphere production under a microscope, the microspheres were collected into cell culture dishes. Step e: Collect the microspheres and cure them under strong ultraviolet irradiation for 30s-300s. Observe the curing of the microspheres under a microscope and measure the diameter of the microspheres (50-250μm). Step f: Elute the corn oil on the surface of the microspheres with 70%-100% ethanol and collect the CsA-GelMA microspheres.

7. A method for preparing a programmable, controllable, sustained-release biomimetic spinal cord graft according to any one of claims 1-6, characterized in that: At 4°C, CsA-GelMA microspheres and grooved micro / nano-oriented fiber scaffolds were mixed. CsA-GelMA microspheres self-assembled on the grooved micro / nano-oriented fiber scaffold to form a scaffold-microsphere morphology. Human umbilical cord mesenchymal stem cell-derived decellularized matrix (HUC-MSCs-ECM) was then wrapped around the scaffold-microspheres, with the scaffold-microsphere morphology acting as an axis. The scaffold-microsphere morphology was thus encapsulated by the human umbilical cord mesenchymal stem cell-derived decellularized matrix (HUC-MSCs-ECM), with the grooved micro / nano-oriented scaffold serving as the core. The spindle is wrapped with 1-10 layers and freeze-dried to prepare the initial form of a programmable, controllable, sustained-release biomimetic spinal cord graft. Exosomes HUC-MSCs-EXO derived from human umbilical cord mesenchymal stem cells are collected using an ion exchange method. After freeze-drying, the initial form of the programmable, controllable, sustained-release biomimetic spinal cord graft is immersed in the extracted HUC-MSCs-EXO solution to adsorb the exosomes HUC-MSCs-EXO derived from human umbilical cord mesenchymal stem cells. The adsorption time of the exosomes derived from human umbilical cord mesenchymal stem cells is 1-12 hours before use, thus forming a programmable, controllable, sustained-release biomimetic spinal cord graft.

8. The application of a programmed controllable sustained-release bionic spinal cord graft as described in any one of claims 1-6 or a programmed controllable sustained-release bionic spinal cord graft prepared by the method described in claim 7 in materials for repairing spinal cord injuries.

9. The application according to claim 8, characterized in that: The grooved micro / nano oriented fiber scaffold is a biodegradable scaffold. It incorporates recombinant NT3, silk fibroin, neurotrophic factor β-NGF, and bovine collagen, exhibiting good biocompatibility. Factors beneficial for spinal cord injury repair are released in a programmed and controllable manner as the scaffold material degrades in vivo, providing a suitable microenvironment for nerve regeneration, guiding nerve axon growth, providing neuroprotection, and promoting nerve regeneration. The GelMA microspheres containing cyclosporine a can programmatically release cyclosporine a, exerting an immunomodulatory effect in the early stages of spinal cord injury. The scaffold-microsphere morphology is encapsulated with decellularized matrix HUC-MSCs-ECM derived from human umbilical cord mesenchymal stem cells, providing a beneficial microenvironment for nerve cell growth, as well as guiding and oriented growth.

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