Conductive nerve repair conduit and method of making same

Abstract

The application belongs to the technical field of biomedical materials and tissue regeneration, and particularly discloses a conductive nerve repair conduit and a preparation method thereof. The application prepares a biodegradable nerve conduit based on silk fibroin, which has good biocompatibility and mechanical properties matched with the mechanical properties of nerves. In-vivo and in-vitro biological experiments show that the conductive conduit can reshape the nerve inflammatory microenvironment in the conduit and indirectly promote the repair of peripheral nerves. By applying electric stimulation, the regeneration of nerve tissue can be better accelerated, and the repair of nerve injury can be completed.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and tissue regeneration technology, specifically to a conductive nerve repair conduit with neuromechanical properties that can reshape the inflammatory microenvironment and its preparation method. Background Technology

[0002] Peripheral nerve injury (PNI) is a common clinical condition that can lead to severe limb disability and impose a significant burden on society. The main reason for the poor prognosis of PNI treatment is the interference of surrounding muscle tissue and the inflammatory environment. Nerve conduits are a primary treatment strategy for PNI, providing ideal mechanical support for nerve regeneration and preventing surrounding tissues from interfering with nerve repair. While nerve conduits can block the interference of extraductal inflammatory factors on nerve regeneration, the inflammatory microenvironment within the nerve conduit, such as the large amounts of inflammatory substances released by Schwann cell pyroptosis, severely impairs peripheral nerve regeneration. The inflammatory microenvironment consists of inflammatory cells, pro-inflammatory enzymes, and inflammatory mediators. At the site of nerve injury, the main inflammatory cells are pro-inflammatory macrophages (M1) and pyroptotic Schwann cells. During nerve injury, Schwann cells undergo pyroptosis and release various inflammatory factors, such as interleukins IL-18 and IL-1β. Simultaneously, M1 macrophages secrete TNF-α, ultimately directly inhibiting peripheral nerve regeneration. Studies have shown that the micropatterns and topology of neural conduits can modulate macrophage polarization, but cannot inhibit Schwann cell pyroptosis or reduce the release of inflammatory factors in the conduits.

[0003] Besides the challenges posed by the inflammatory microenvironment, nerve conduits also need to overcome the problem of mechanical property mismatch. Recently, silk fibroin (RSF)-based materials have been widely used in regenerative medicine due to their significant biocompatibility, such as in cartilage, nerve, and corneal repair. Currently, silk fibroin-based conduits have been manufactured for nerve and urethral repair, but their therapeutic efficacy remains unsatisfactory due to mechanical property mismatch. Conduits used for peripheral nerve regeneration should have an elastic modulus matching that of the peripheral nerve and a high elongation at break. Both high and low elastic moduli are detrimental to peripheral nerve regeneration. The elastic modulus of the human sciatic nerve is approximately 6.71 ± 0.93 MPa. Some silk fibroin-based conduits with elastic moduli reaching several hundred MPa are stiffer than the nerve, potentially trapping it or even hindering regeneration. Simultaneously, some silk fibroin-based conduits with very low elastic moduli cannot support peripheral nerve regeneration and may collapse after application, causing surrounding muscles to compress the nerve and hindering regeneration. Furthermore, low elongation at break may cause the conduit to break and fall during movement, making it difficult to meet the requirements for long-term use.

[0004] In addition, electrical stimulation (ES) can treat peripheral nerve injuries by promoting the extension of nerve axons and modulating macrophage polarization. Currently, adding conductive substances such as polypyrrole, polyaniline, and polythiophene can enhance the electrical conduction of catheters; however, their biocompatibility remains unsatisfactory. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a conductive nerve repair catheter and its preparation method. The conductive nerve repair catheter is an easily fabricated DMF / RSF / P:P composite catheter that possesses high elasticity and sustained-release drug properties, while also exhibiting mechanical properties that match neuromechanical properties.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0007] A first aspect of the present invention is to provide a method for preparing a conductive nerve repair conduit, specifically comprising the following steps:

[0008] Step 1: Prepare regenerated silk protein stock solution;

[0009] Step 2: Take the regenerated silk protein stock solution obtained in Step 1, mix it with poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) aqueous solution (PEDOT:PSS) and dimethyl fumarate (DMF), crosslink it with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), and prepare a film by the saliva method.

[0010] Step 3: After the film has formed, soak it in an ethanol solution to obtain better flexibility and make it easy to roll into a conduit.

[0011] In the preparation method described above, DMF plays a role in regulating the inflammatory microenvironment. DMF can promote the polarization of macrophages towards the M2 subtype and inhibit pyroptosis by targeting Gasdermin D (GSDMD). Therefore, the local application of the DMF release system can be an effective supplement to the nerve conduit to regulate the inflammatory microenvironment. PEDOT:PSS is an excellent metal and inorganic semiconductor material with excellent conductivity and biocompatibility. It exhibits good stability in various environments and can better enhance the effect of electrical stimulation.

[0012] Preferably, the process of preparing regenerated silk protein stock solution in step 1 is as follows: natural mulberry cocoons or silkworm silk are degummed and dried to produce degummed silk. The degummed silk is dissolved, and then impurities are removed by dialysis and concentrated to obtain regenerated silk protein stock solution.

[0013] Furthermore, the degumming is performed using a sodium carbonate solution; the dissolution is performed using a lithium bromide solution; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 14,000; and the concentration is performed using a high molecular weight polymer for dialysis concentration.

[0014] Furthermore, the polymer is polyethylene glycol with a molecular weight of 6000 to 12000.

[0015] Preferably, in step 2, the concentration of the regenerated silk protein stock solution is 4 wt%. Too high a concentration of the regenerated silk protein stock solution will cause a mismatch with the elastic modulus of nerve fibers, while too low a concentration will make film formation difficult.

[0016] Preferably, in step 2, the concentration of horseradish peroxidase is 180–200 U / mL, and the concentration of hydrogen peroxide is 0.4–0.6 wt%.

[0017] Preferably, in step 2, the concentration of PEDOT:PSS is 0.2% w / v, and the concentration of DMF is 400 μM. Excessive concentrations of either can affect cell viability and even cause cell death. Insufficient PEDOT:PSS concentration leads to poor conductivity. Insufficient DMF concentration weakens the membrane material's ability to inhibit pyroptosis.

[0018] A second aspect of this invention is to provide a conductive nerve repair conduit prepared according to the above-described method. The inventors have demonstrated through evaluation experiments that the conductive nerve repair conduit exhibits excellent cell compatibility and inhibits Schwann cell pyroptosis, thereby reshaping the inflammatory microenvironment within the conduit at the site of injury and promoting the recovery of damaged nerves.

[0019] A third aspect of this invention is the application of the conductive nerve repair catheter prepared according to the above method in the preparation of products for peripheral nerve injury repair. In practical application, the conductive nerve repair catheter is sutured to the epineurium using 10-0 monofilament nylon sutures, and electrical stimulation can better repair nerve damage. The inventors applied the DMF / RSF / P:P catheter and electrical stimulation to a Sprague-Dawley (SD) rat sciatic nerve injury model for evaluation and obtained very good damage repair results.

[0020] The present invention has the following beneficial effects:

[0021] This invention utilizes RSF as the matrix, PEDOT:PSS as the conductive material, DMF as an inflammation inhibitor, and HRP and H2O2 as chemical cross-linking agents to prepare a composite catheter. This composite catheter exhibits good biocompatibility, can remodel the inflammatory microenvironment, and possesses mechanical properties that match neuromechanical characteristics, thus improving the repair effect of nerve tissue and achieving rapid repair and regeneration of nerve damage. Furthermore, the preparation method of this invention is simple and easy to implement, and readily applicable. Attached Figure Description

[0022] Figure 1 : Schematic diagram of the external structure of the conductive nerve repair catheter of the present invention.

[0023] Figure 2 : Flowchart of the preparation process of the conductive nerve repair conduit of the present invention.

[0024] Figure 3 Microscopic morphology of the conductive nerve repair conduit in Example 1.

[0025] Figure 4 Biocompatibility results of the conductive nerve repair conduit in Example 2. In the figure, from left to right, Day 1 and Day 7 represent the live cell view, dead cell view, and overlay view, respectively.

[0026] Figure 5 Transmission electron microscopy image of Schwann cell growth on the conductive nerve repair conduit in Example 2.

[0027] Figure 6 Example 3: An animal model for assessing sciatic nerve injury in rats using a conductive nerve repair catheter.

[0028] Figure 7 : In vivo sciatic nerve index recovery diagram of the conductive nerve repair catheter in Example 3.

[0029] Figure 8 Mechanical data of the conductive nerve repair conduit in Example 4. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1: Preparation of conductive nerve repair conduit

[0032] A conductive nerve repair conduit, such as Figure 1 As shown, its preparation process is as follows: Figure 2 As shown, the specific preparation method is as follows:

[0033] 1. Boil the silk in a 0.02M Na₂CO₃ solution at 100℃ for 30 minutes, then wash with distilled water and dry in an incubator at 37℃ for 3 days. The extracted silk fibroin is dissolved in a 9.3 mol / L LiBr solution at 60℃. –1 The solution was then dialyzed against deionized water over 1 hour. Subsequently, the prepared solution was dialyzed against deionized water on a dialysis membrane for 3 days at room temperature. Finally, the RSF solution was concentrated with polyethylene glycol (PEG) solution for 1 day to obtain the silk fibroin RSF solution.

[0034] 2. PEDOT:PSS aqueous solution and DMF solution were added to the silk fibroin RSF solution to achieve the final target concentration. Subsequently, equal volumes of horseradish peroxidase (HRP) solution and hydrogen peroxide (H2O2) solution (0.3% v / v) were added to the mixed solution at a volume ratio of 1:25 to promote cross-linking. After thorough mixing, the solution was poured into a flat-bottomed polystyrene well plate and allowed to air dry to obtain a conductive film.

[0035] 3. Dry at room temperature overnight, then immerse the conductive film in ethanol (75% v / v) solution for 6 hours to make it insoluble in water and easy to roll into a conduit.

[0036] The DMF / RSF / P:P catheters were immersed in a poly-L-lysine solution (0.1% w / v) for 2 hours to promote cell adhesion in in vivo and in vitro experiments.

[0037] More specifically, in step 2 of this embodiment, the concentration of PEDOT:PSS was 0.2% w / v, the concentration of DMF was 400 μM, the concentration of horseradish peroxidase was 200 U / mL, the concentration of hydrogen peroxide was 0.4 wt%, and the RSF solution was tested at three concentrations: 4%, 8%, and 12%.

[0038] The microstructure of the fabricated conductive nerve repair conduit is as follows: Figure 3 As shown, the surface roughness of the film material is related to the concentration of RSF; that is, the surface roughness of RSF decreases with increasing RSF concentration. The film surfaces of the RSF-only group and the DMF / 4wt%RSF / P:P group are uneven, which can promote cell adhesion, while the film surfaces of the DMF / 8wt%RSF / P:P and DMF / 12wt%RSF / P:P groups are relatively smooth (the results of 12wt%RSF are similar to those of 8wt%RSF, therefore...). Figure 3 (Not given in the text).

[0039] Example 2: In vitro experiment of conductive nerve repair conduit

[0040] 1. A DMF / RSF / P:P composite membrane was prepared according to the method in Example 1. In step 2, the concentration of DMF was 400 μM, the concentration of horseradish peroxidase was 200 U / mL, the concentration of hydrogen peroxide was 0.4 wt%, the concentration of RSF solution was 4%, and three concentrations of PEDOT:PSS were tested: 0.1%, 0.2%, and 0.3%.

[0041] After sterilizing the prepared DMF / RSF / P:P composite membrane by ultraviolet irradiation and alcohol soaking, Schwann cells were seeded on it, and the proliferation of cells on the surface of the composite membrane was observed by staining with live and dead cells.

[0042] The results are as follows Figure 4 As shown, when the PEDOT:PSS concentration was 0.3% w / v, the number of dead cells increased significantly on days 1 and 7.

[0043] 2. A DMF / RSF / P:P composite membrane was prepared according to the method in Example 1. In step 2, the concentration of PEDOT:PSS was 0.2% w / v, the concentration of DMF was 400 μM, the concentration of horseradish peroxidase was 200 U / mL, the concentration of hydrogen peroxide was 0.4 wt%, and the concentration of RSF solution was 4 wt%.

[0044] After sterilization by ultraviolet irradiation and alcohol immersion, the prepared DMF / RSF / P:P composite membrane was seeded with Schwann cells, and their growth on the composite membrane surface was observed by transmission electron microscopy. Experiments were conducted in PBS, DMF, ES, and DMF / ES groups by controlling two variables: DMF and electrical stimulation. The results are as follows: Figure 5 As shown, compared with the PBS group where cells exhibited pyroptosis, the cells in the other groups had normal morphology and intact structure.

[0045] Example 3: In vivo experiment of conductive nerve repair conduit

[0046] 1. After anesthesia, the left hind leg of rats was shaved and disinfected in preparation for surgery. A clamp model of the sciatic nerve was created by clamping it with hemostatic forceps fitted with a plastic tube. In the sham surgery group, the sciatic nerve was exposed but not damaged; the skin and muscle layers were then sutured together. In the nerve injury group, a nerve sample was obtained 1 cm distal to the site of nerve injury, and nerve samples of the same length were also collected in this group. All surgeries were performed by the same operator. Before implantation, the "membrane" was softened by soaking in PBS solution, making it easy to roll into a conduit. The membrane was then wrapped around the sciatic nerve and secured to the epineurium with 10-0 monofilament nylon suture, as shown. Figure 6 As shown. The skin and muscle layers were then sutured, and subsequent experiments continued. Animals in the electrical stimulation group received 30 minutes of electrical stimulation (7mA) daily for two weeks post-surgery. In short, rats were placed in a fixation device with two surface electrodes placed on the skin above the gastrocnemius muscle of the left leg and connected to a stimulator. The optimal current parameters for electrical stimulation were based on previous literature reports and preliminary experiments by the research group, namely, the lowest current value that would induce visible foot and toe movements in rats during electrical stimulation without causing significant pain or discomfort. In this embodiment, to investigate the effects of DMF and electrical stimulation on the recovery of nerve function, four groups were set up for the nerve injury group: no DMF, no electrical stimulation, both, and neither.

[0047] 2. The effect of conductive materials combined with electrical stimulation on the recovery of motor function after sciatic nerve injury was evaluated by analyzing the gait trajectories of rats at different time points. At 4, 8, and 12 weeks post-surgery, rats were dipped in black ink in their hind paws and allowed to walk along a narrow path, leaving footprints on white paper. The SFI (Sciatic Nerve Function Index) was calculated using the following formula: SFI = -38.3 × (EPL - NPL) / NPL + 109.5 × (ETS - NTS) / NTS + 13.3 × (EIT - NIT) / NIT - 8.8. PL is the distance from the heel to the third toe, TS is the distance from the first to the fifth toe, and IT is the distance from the second to the fourth toe. E and N represent the experimental group and the normal group, respectively. The final results were then statistically analyzed, with 6 rats measured in each group.

[0048] The results are as follows Figure 7 As shown, rats treated with added DMF and electrical stimulation had higher sciatic nerve function indices and better recovery. Both DMF and electrical stimulation had significant effects on the sciatic nerve function index, with DMF having a greater effect than electrical stimulation.

[0049] Example 4: Mechanical property testing of conductive nerve repair conduit

[0050] The mechanical properties of the DMF / RSF / P:P membrane prepared according to the method of Example 1 were tested using an MTS CMT6103 mechanical testing instrument. The mechanical properties of the membrane were tested in two states: wet and dry. Dry state refers to drying the membrane at room temperature for 48 hours before testing; in contrast, wet state refers to immersing the membrane in deionized water for 24 hours before testing. In the tensile test, the hydrogel sample was a strip 50 mm × 10 mm × 1 mm in shape, with a clamp spacing of 20 mm and a tensile rate of 15 mm·min. -1 Each sample group should be tested at least 5 times, and the values ​​are expressed as mean ± SD.

[0051] The results are as follows Figure 8 As shown, the elastic modulus of the conductive nerve repair conduit is very close to that of a real nerve, indicating a high degree of matching.

[0052] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A method for preparing a conductive nerve repair conduit, characterized in that: Includes the following steps: Step 1: Prepare regenerated silk protein stock solution; Step 2: Take the regenerated silk protein stock solution obtained in Step 1, mix it with PEDOT:PSS aqueous solution and DMF solution, add horseradish peroxidase and hydrogen peroxide for cross-linking, and then prepare a film by casting method; the concentration of the regenerated silk protein stock solution is 4wt%, the concentration of PEDOT:PSS is 0.2% w / v, and the concentration of DMF is 400μM. Step 3: After the film has formed, soak it in an ethanol solution to obtain better flexibility and make it easy to roll into a conduit.

2. The method for preparing the conductive nerve repair conduit according to claim 1, characterized in that: The process of preparing regenerated silk protein stock solution in step 1 is as follows: natural mulberry cocoons or silkworm silk are degummed and dried to make degummed silk. The degummed silk is dissolved, and then impurities are removed by dialysis and concentrated to obtain regenerated silk protein stock solution.

3. The method for preparing the conductive nerve repair conduit according to claim 2, characterized in that: The degumming is performed using a sodium carbonate solution; the dissolution is performed using a lithium bromide solution; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 14,000; and the concentration is performed using a high molecular weight polymer for dialysis concentration.

4. The method for preparing the conductive nerve repair conduit according to claim 3, characterized in that: The polymer is polyethylene glycol with a molecular weight of 6000-12000.

5. The method for preparing the conductive nerve repair conduit according to claim 1, characterized in that: In step 2, the concentration of horseradish peroxidase is 180~200 U / mL, and the concentration of hydrogen peroxide is 0.4~0.6 wt%.

6. The conductive nerve repair conduit prepared by the method according to any one of claims 1-5.

7. The use of the conductive nerve repair catheter according to claim 6 in the preparation of products for peripheral nerve injury repair.

Images