Nerve grafts, methods of preparation and use

Abstract

The application discloses a nerve graft, a preparation method and application, and relates to a nerve conduit with an inner wall loaded with activin A-containing microspheres. The nerve graft provided by the application is a growth factor delivery system with excellent performance and controllable release kinetics, can realize efficient repair of a nerve defect of a rat, and the preparation method of the nerve graft is convenient and easy to operate, thereby laying a reliable technical foundation for development of a next-generation intelligent nerve graft.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and more particularly to a neural graft, its preparation method, and its application. Background Technology

[0002] Nerve injury is a common clinical challenge, caused by factors such as traffic accidents, work-related injuries, or iatrogenic injuries. It often leads to impairment of motor, sensory, and autonomic nervous system functions, severely impacting patients' quality of life. Based on the degree of damage to the neuroanatomical structure, it can be classified into different types. Among them, neurotmesis is the most severe form of injury, referring to a complete rupture of the nerve trunk, sometimes accompanied by defects, resulting in a complete loss of nerve continuity. In this case, Wallerian degeneration occurs in the nerve distal to the injury. Due to the interruption of the endoneurotic canal, regenerating axons cannot accurately ingrow into the distal segment, often forming neuromas, making spontaneous functional recovery impossible.

[0003] Currently, the gold standard for clinical treatment remains autologous nerve transplantation, which involves harvesting a secondary nerve (such as the sural nerve) from the patient's own body to bridge the defect. While this method provides an ideal regenerative microenvironment, its application is limited by factors such as limited donor sites, potential secondary donor site damage, and possible size mismatches. For cases with no defects or low tension at the severed ends, direct suturing may be used; however, if a defect exists, direct suturing can create tension, negatively impacting blood supply and regeneration. Furthermore, clinical practice guidelines indicate that for nerve transection, conservative treatment or rehabilitation alone is often ineffective, frequently requiring surgical intervention.

[0004] To overcome these limitations, tissue-engineered neural grafts have emerged, utilizing biocompatible scaffolds to mimic the structure of the neural basilar membrane tubules, providing physical channels and guidance for axonal regeneration. Ideally, grafts should be loaded with active factors to create a dynamic microenvironment that promotes regeneration locally. However, on the one hand, the selection of suitable active factors is still limited; on the other hand, simply adsorbed active factors often experience rapid loss due to the "burst release effect," failing to maintain an effective concentration at the site of injury and severely limiting repair efficacy. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a nerve graft that can be used for efficient repair of nerve injuries, and the second purpose is to provide a method for preparing and applying the nerve graft.

[0006] Technical solution: The neural graft of the present invention is a neural conduit with activin A microspheres loaded on its inner wall.

[0007] Preferably, the microspheres containing activator A are made of gelatin, methacrylamide gelatin, collagen, chitosan, hyaluronic acid, or polylactic acid-glycolic acid copolymer, and are one or more of the following materials that adsorb activator A recombinant protein: gelatin, methacrylamide gelatin, collagen, chitosan, hyaluronic acid, or polylactic acid-glycolic acid copolymer.

[0008] Preferably, the nerve conduit is a conduit made of silk fibroin and / or collagen.

[0009] The method for preparing the neural graft according to the present invention includes the following steps: (1) Using a photoinitiator-containing methacrylamide gelatin solution as the dispersed phase and an oil phase solvent as the continuous phase, W / O type droplets were prepared using a microfluidic chip device. After ultraviolet irradiation, the droplets were washed and freeze-dried to obtain microspheres. (2) The microspheres obtained in step 1 were incubated in a recombinant protein solution containing activin A to obtain microspheres containing activin A; (3) The collagen solution was added dropwise to the silk fibroin solution, injected into the mold and then freeze-dried. After cross-linking, a nerve conduit was obtained. (4) Load the microspheres containing activin A obtained in step 2 into the neural conduit obtained in step 3 to obtain a neural graft.

[0010] Preferably, in step 1, the concentration of methacrylamide gelatin is 3~10% w / v; the concentration of photoinitiator is 0.01~0.1% w / v; and the oil phase solvent is corn oil.

[0011] Preferably, in step 1, the microfluidic chip device assembly steps are as follows: a) Capillary preparation: Select glass capillaries with an inner diameter of 560-600 μm and an outer diameter of 1 mm as the outer phase tube; simultaneously, prepare the inner phase tube using glass capillaries of the same specifications, drawing one end into a tapered shape using a microelectrode drawing instrument, and then polishing the tip of the tapered shape with fine sandpaper to an inner diameter of 80-200 μm. Also select square glass capillaries with an inner diameter of 1.05 mm as the sleeve. All glass capillaries are cleaned, dried, and sterilized before use.

[0012] b) Chip Assembly: Inside the clean bench, place a clean glass slide as the substrate. Use AB glue to horizontally fix the sleeve in the center of the slide. After the glue has cured, first insert and position the outer phase tube from one end of the sleeve. Then, coaxially insert the inner phase tube (with the conical end facing the outlet) into the outer phase capillary tube, finely adjusting its position so that the conical port of the inner phase tube is located in the center inside the port of the outer phase tube. Select a micro-sampling needle and fix it above the interface between the inner phase tube and the sleeve as the phase inlet; use AB glue to seal all positions except the inner phase inlet, outer phase inlet, and outlet to ensure the device is leak-free.

[0013] Preferably, in step 1, when using a microfluidic chip device for preparation, the continuous phase flow rate is 15~60 mL / h, the dispersed phase flow rate is 0.5~3 mL / h, and the flow rate ratio of the continuous phase to the dispersed phase is 20:1~60:1.

[0014] Preferably, in step 2, the concentration of the activin A recombinant protein solution is 0.1~10 ng / μL.

[0015] Preferably, in step 3, the crosslinking step includes physical crosslinking by immersion in ethanol and chemical crosslinking by immersion in a crosslinking agent, wherein the crosslinking agent is a morpholine ethanesulfonic acid buffer containing carbodiimide hydrochloride and N-hydroxysuccinimide, with a pH of 5.5 to 6.7, and the mass ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1 to 2:1.

[0016] Preferably, in step 4, the loading method is to coat or inject a microsphere suspension containing activator A.

[0017] The application of the nerve graft described in this invention in the preparation of medical devices for the treatment of nerve injuries.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the neural graft loaded with activin A microspheres provides a high-performance growth factor delivery system that can achieve efficient repair of nerve defects in rats, and the preparation method of the neural graft is convenient and easy to operate, laying a reliable technical foundation for the development of next-generation intelligent neural grafts. Attached Figure Description

[0019] Figure 1 Schematic diagram of a single emulsion droplet microfluidic device; Figure 2 This is a flowchart illustrating the preparation process of microspheres containing activator A. Figure 3 A schematic diagram of a nerve graft loaded with activin A microspheres for bridging sciatic nerve injury; Figure 4 The image shows the results of using a nerve graft loaded with activin A microspheres for the repair of sciatic nerve injury. The scale bar is 1000 μm. Detailed Implementation

[0020] The technical solution of the present invention will be further described below.

[0021] Example 1: Preparation of neural grafts loaded with activin A microspheres 1. Solution preparation a) Dispersed phase: Under aseptic conditions, weigh 0.05 g of methacrylamide gelatin solid powder (GelMA, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., product number EFL-GM-60), dissolve it in 1 mL of sterile phosphate buffer (PBS), heat in a 50°C water bath and stir until completely dissolved, centrifuge to remove air bubbles to obtain a clear, bubble-free 5% (w / v) GelMA solution, and keep it at 37°C for later use.

[0022] Add 1% volume of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP, CAS No. 85073-19-4) solution (10% w / v) to the GelMA solution, vortex mix, filter using a 0.22 μm sterile filter, and store in the dark for later use.

[0023] b) Continuous phase: The corn oil (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number C116025-500ml) was filtered through a 0.22 μm sterile filter and then used for later use.

[0024] 2. Preparation of a single emulsion droplet microfluidic device a) Capillary preparation: A glass capillary with an inner diameter of 580 μm and an outer diameter of 1 mm was selected as the outer phase tube. Simultaneously, an inner phase tube was made from a glass capillary of the same specifications. One end of the inner phase tube was drawn into a tapered shape using a microelectrode drawing instrument, and then the tip of the tapered shape was polished with fine sandpaper to an inner diameter of approximately 120 μm. A square glass capillary with an inner diameter of 1.05 mm was also selected as the sleeve. All glass capillary tubes were cleaned, dried, and sterilized before use.

[0025] b) Chip Assembly: Inside the laminar flow hood, place a clean glass slide as the substrate. Use AB glue to horizontally fix the sleeve in the center of the slide. After the glue cures, first insert and position the outer phase tube from one end of the sleeve. Then, coaxially insert the inner phase tube (with the conical end facing the outlet direction) into the outer phase capillary tube, finely adjusting its position so that the conical end of the inner phase tube is centered inside the outer phase tube end. Select a micro-sampling needle and fix it above the interface between the inner phase tube and the sleeve as the outer phase inlet; use AB glue to seal all locations except the inner phase inlet, outer phase inlet, and outlet to ensure the device is leak-free. Figure 1 As shown.

[0026] After assembly, use an alcohol lamp flame or a miniature blowtorch to quickly scorch the inlet end of the inner phase tube and the outlet end of the outer phase tube to make the tube openings smooth.

[0027] 3. Preparation of microspheres The dispersed phase prepared in step 1 is loaded into a 1 mL sterile syringe, and the continuous phase is loaded into a 5 mL sterile syringe. These are then installed in separate syringe pumps and connected to the inner and outer phase inlets of the microfluidic chip via sterile polyethylene tubing.

[0028] Turn on the continuous phase injection pump at a flow rate of 30 mL / h. After observing a stable oil flow at the outlet under a microscope, turn on the dispersed phase injection pump at a flow rate of 1 mL / h. At this point, stable and uniform W / O droplets are observed forming at the port of the inner phase tube under a microscope. After the droplet formation stabilizes, begin collecting the outflowing emulsion into sterile cell culture dishes.

[0029] One hour after droplet collection, the culture dish was immediately placed under a 365 nm ultraviolet light source and irradiated for 180 seconds to induce photocrosslinking of the LAP-GelMA droplets, which solidified into spheres. The upper oil phase was carefully removed, and the microspheres were cleaned with anhydrous ethanol to remove residual corn oil from the surface.

[0030] After washing, the microspheres were transferred to sterile, enzyme-free centrifuge tubes. After the anhydrous ethanol evaporated naturally, 1.2 mL of PBS was added to fully rehydrate and swell the microspheres. Excess PBS was removed, and the microspheres were aliquoted into centrifuge tubes at 45 μL per tube. The rehydrated microspheres were then freeze-dried at -80 °C for 24 h to obtain GelMA microsphere powder, which was then dried and stored at -20 °C.

[0031] 4. Preparation of microspheres containing activator A A solution of recombinant activin A protein (purchased from Abcam, catalog number ab151687) at a concentration of 0.166 ng / μL was prepared using sterile PBS.

[0032] Take one vial of the freeze-dried GelMA microspheres prepared in step 3, add 50 μL of a 0.166 ng / μL solution of recombinant activin A protein (purchased from Abcam, catalog number ab151687) prepared with sterile PBS, and incubate with gentle shaking at 4°C for 2 h to allow the microspheres to fully rehydrate and adsorb activin A, ensuring adsorption equilibrium. After adsorption is complete, microspheres containing activin A are obtained.

[0033] The preparation process of microspheres containing activator A is as follows: Figure 2 As shown.

[0034] 5. Preparation of nerve grafts Bovine type I collagen (purchased from Guangzhou Chuang'er Biotechnology Co., Ltd., catalog number YC0012) was dissolved in 2% acetic acid solution to prepare a 1 wt% collagen solution. Regenerated silk fibroin was prepared according to the method described in the reference (DOI:10.1016 / j.colsurfb.2018.10.053), and then dissolved in deionized water to prepare a silk fibroin solution. At room temperature, 1 mL of collagen solution was added dropwise to a 10 wt% silk fibroin solution at a rate of 2 seconds / drop and stirred at 800 rpm for 4 h to obtain a silk fibroin / collagen solution. This solution was then injected into a hollow tubular mold with an inner diameter of 2 mm, an outer diameter of 3 mm, and a length of 30 mm. The mold was then frozen at -20℃ and freeze-dried (cold trap temperature -80℃, duration 24 h). Subsequently, the mold was immersed in ethanol for 24 h for physical cross-linking, and then freeze-dried again (cold trap temperature -80℃, freeze-drying for 24 h). Finally, the mold was sealed in a ternary cross-linking agent and immersed at room temperature for 24 h. h, a silk fibroin / collagen composite duct was obtained, wherein the ternary cross-linking agent was a 2-morpholine ethanesulfonic acid buffer containing 4% (w / v) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E106172-100g) and 2% (w / v) N-hydroxysuccinimide (purchased from Absin, catalog number abs45156723-25g). After cross-linking, it was taken out and washed three times with 50 mL of deionized water, each time for 0.5 h. Then it was placed in a -20℃ freezer for 6 h, and then freeze-dried for 24 h. After drying, 12 mm lengths were cut for later use.

[0035] Take 45 μL of the above-mentioned activin A-containing microspheres and dilute them with 555 μL of PBS to obtain a microsphere suspension. Inject 40 μL of the microsphere suspension into the inner wall of a 12 mm long silk fibroin bovine collagen catheter. After the solution is completely absorbed, a nerve graft loaded with activin A microspheres is obtained and stored at 4°C for later use.

[0036] Example 2: Treatment of a rat model of sciatic nerve defect using a neural graft loaded with activin A microspheres. Eight-week-old male SD rats weighing 180-220g were anesthetized and their skin was prepared. The sciatic nerve in the left hind limb of the rat was separated and transversely cut to form a 10 mm gap, thus constructing a rat model of sciatic nerve defect in the left hind limb. After modeling, all rats were randomly divided into experimental group and control group, with 10 rats in each group.

[0037] In the experimental group, absorbable surgical sutures were used to microsurgically anastomose the two ends of a nerve graft loaded with activin A microspheres to the severed nerve ends, bridging and repairing sciatic nerve defects in rats (e.g., Figure 3 (as shown in the figure) to evaluate its effectiveness in repairing long-distance peripheral nerve defects; the control group only used silk fibroin / collagen composite catheters for bridging.

[0038] Three weeks after injury, sciatic nerve tissue was collected, frozen sectioned, and blocked with immunostaining blocking solution for 50 min. It was then incubated overnight at 4°C with 1:500 diluted SCG10 primary antibody (Novus, catalog number NBP1-49461). After incubation, the tissue was washed with PBS buffer and then incubated with 1:400 diluted Alexa Fluor. TM The 488 fluorescent secondary antibody (purchased from Invitrogen, catalog number A-21206) was incubated at room temperature in the dark. After incubation, the cells were rinsed and counterstained with DAPI. The cells were then observed and images were acquired using a fluorescence microscope.

[0039] The results are as follows Figure 4 As shown, compared with the control group, the length of SCG10-labeled regenerated nerve fibers in the experimental group was significantly increased, indicating that the tissue-engineered nerve graft can effectively promote axonal regeneration after sciatic nerve injury.

Claims

1. A neural graft, characterized in that, The neural graft is a neural conduit with an inner wall loaded with activin A microspheres.

2. The neural graft according to claim 1, characterized in that, The microspheres containing activator A are made of gelatin, methacrylamide gelatin, collagen, chitosan, hyaluronic acid, or polylactic acid-glycolic acid copolymer, and are used to adsorb recombinant activator A protein.

3. The neural graft according to claim 1, characterized in that, The nerve conduit is a conduit made of silk fibroin and / or collagen.

4. A method for preparing a neural graft according to any one of claims 1 to 3, characterized in that the steps include... include: (1) Using a photoinitiator-containing methacrylamide gelatin solution as the dispersed phase and an oil phase solvent as the continuous phase, W / O type droplets were prepared using a microfluidic chip device. After ultraviolet irradiation, the droplets were washed and freeze-dried to obtain microspheres. (2) The microspheres obtained in step 1 were incubated in a recombinant protein solution containing activin A to obtain microspheres containing activin A; (3) The collagen solution was added dropwise to the silk fibroin solution, injected into the mold and then freeze-dried. After cross-linking, a nerve conduit was obtained. (4) Load the microspheres containing activin A obtained in step 2 into the neural conduit obtained in step 3 to obtain a neural graft.

5. The preparation method according to claim 4, characterized in that, In step 1, the concentration of the methacrylamide gelatin is 3-10% w / v; the concentration of the photoinitiator is 0.01-0.1% w / v; and the oil phase solvent is corn oil.

6. The preparation method according to claim 4, characterized in that, In step 1, the inner diameter of the outlet end of the dispersed phase tube in the microfluidic chip device is 80~200 μm; during preparation, the continuous phase flow rate is 15~60 mL / h, the dispersed phase flow rate is 0.5~3 mL / h, and the flow rate ratio of the continuous phase to the dispersed phase is 20:1~60:

1.

7. The preparation method according to claim 4, characterized in that, In step 2, the concentration of the activin A recombinant protein solution is 0.1~10 ng / μL.

8. The preparation method according to claim 4, characterized in that, In step 3, the crosslinking step includes physical crosslinking by immersion in ethanol and chemical crosslinking by immersion in a crosslinking agent, wherein the crosslinking agent is a morpholine ethanesulfonic acid buffer containing carbodiimide hydrochloride and N-hydroxysuccinimide, with a pH of 5.5~6.7 and a mass ratio of carbodiimide hydrochloride to N-hydroxysuccinimide of 1~2:

1.

9. The preparation method according to claim 4, characterized in that, In step 4, the loading method is to coat or inject a microsphere suspension containing activator A.

10. The use of a nerve graft according to any one of claims 1 to 3 in the preparation of a medical device for the treatment of nerve injury.

Images