Nerve conduit for nerve injury repair and preparation method thereof

Abstract

The invention provides a nerve conduit for nerve injury repair and a preparation method thereof, and belongs to the technical field of medical materials. The nerve conduit provided by the invention is composed of three-dimensional conductive composite hydrogel loaded with human endometrial mesenchymal stem cells, and conductive polymer polypyrrole is grafted and crosslinked in a natural biological material network formed by chitosan and gelatin. The catheter has good biocompatibility, degradability and stable conductivity, can simulate an electrophysiological microenvironment for nerve regeneration, and can generate synergy with a paracrine function of stem cells, so that the directional regeneration of axons is effectively guided, and the formation of myelin sheaths is promoted. In-vivo and in-vitro experiments show that when the catheter is used for repairing peripheral nerve long segment defects, the function recovery effect of the catheter is equivalent to that of autologous nerve transplantation, and an ideal alternative scheme is provided for clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to a nerve conduit for nerve injury repair and a preparation method thereof. BACKGROUND

[0002] Functional reconstruction after peripheral nerve injury (PNI) is a serious challenge in the field of clinical medicine and regenerative medicine. Long-segment nerve defects often result in the absence of a regenerative microenvironment and the interruption of guidance cues, making it difficult for neurons to effectively extend, target growth and functional connection, ultimately causing persistent sensory and motor dysfunction in patients.

[0003] To address this problem, nerve guidance conduit (NGC) as an implanted medical device designed to bridge defects and guide regeneration has been widely studied. Ideally, nerve guidance conduit should not only serve as a passive physical channel to isolate the surrounding tissue interference and prevent scar invasion, but also actively create a functional space that can support and guide precise nerve axon regeneration. In the prior art, most nerve guidance conduits focus on improving the biocompatibility of materials, geometric design of macrostructure (such as single / multi-lumen conduit) or introducing simple biochemical modification (such as coating) to provide basic support and limited biological activity.

[0004] However, these strategies are still limited in terms of rebuilding a highly biomimetic, dynamically responsive and coordinated with host nerve regeneration process complex functional microenvironment. In particular, for long-segment defects, existing conduits often have difficulty in achieving deep integration with host nerve tissue in structure and function after implantation, and also have difficulty in providing a comprehensive microenvironment sufficient to drive and guide axon directional growth, myelination and ultimately functional connection in the defect area, which directly restricts its repair effect and application potential in complex clinical scenarios. SUMMARY

[0005] The purpose of the present application is to provide a nerve conduit for nerve injury repair and a preparation method thereof, which solves the problem of limited effect of existing materials in repairing long-segment nerve defects.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides a nerve conduit, comprising a three-dimensional conductive composite hydrogel loaded with human endometrial mesenchymal stem cells, the composite hydrogel comprising a conductive polymer and a biological material, and the conductive polymer being grafted onto the biological material and cross-linked to form a three-dimensional network.

[0008] Preferably, the conductive polymer is polypyrrole, and the biomaterial comprises chitosan and gelatin.

[0009] The present application also provides a preparation method of the nerve conduit, comprising the following steps: S1. dissolving chitosan in an acetic acid-containing solution, adding pyrrole monomers and an oxidizing agent to polymerize the pyrrole monomers, and obtaining a polypyrrole-chitosan composite solution; S2. mixing the polypyrrole-chitosan composite solution with gelatin in the presence of a crosslinking agent, performing a crosslinking reaction, and forming a polypyrrole-chitosan-gelatin hydrogel; S3. loading human endometrial mesenchymal stem cells into the polypyrrole-chitosan-gelatin hydrogel, and obtaining the nerve conduit.

[0010] Preferably, in step S1, the volume concentration of acetic acid in the acetic acid-containing solution is 0.5% to 2%. In step S1, before adding the oxidizing agent, the mixed system containing chitosan and pyrrole monomers is treated in the dark; In step S1, the oxidizing agent is added dropwise into the acetic acid-containing solution at a rate of 0.1 mL / h to 2 mL / h; In step S1, the reaction of polymerizing the pyrrole monomers is performed in the dark for 6 to 72 hours; In step S1, after obtaining the polypyrrole-chitosan composite solution, a step of dialysis purification is further included, the dialysis purification is performed in a phosphate buffer using a dialysis bag with a molecular weight cut-off of 8 kDa to 16 kDa, and the dialysis liquid is replaced 2 to 4 times within 4 to 12 hours; In step S1, before the crosslinking reaction, a step of adjusting the pH of the polypyrrole-chitosan composite solution to 6.0 to 7.0 is further included.

[0011] Preferably, in step S2, the crosslinking reaction is initiated by vortex mixing for 10 to 120 seconds.

[0012] Preferably, in step S1, the concentration of chitosan in the acetic acid-containing solution is 0.5 to 3% by weight. In step S1, the mass ratio of the pyrrole monomers to chitosan is 0.05:1 to 0.5:1.

[0013] Preferably, in step S1, the oxidizing agent is at least one selected from ferric chloride, ammonium persulfate, and hydrogen peroxide.

[0014] Preferably, the oxidizing agent is ferric chloride, and the molar ratio of the ferric chloride to the pyrrole monomers is 0.5:1 to 2:1.

[0015] Preferably, in step S2, the crosslinking agent is selected from at least one of glutaraldehyde, genipin, and a combination of carbodiimide and N-hydroxysuccinimide. In step S3, the seeding density of the human endometrial mesenchymal stem cells used is 0.5*10^6 to 2*10^6 cells / mL. In step S3, after the stem cells are loaded on the hydrogel, a step of co-incubation for 4 to 24 hours is further included.

[0016] The application also provides a use of the nerve conduit as described above or prepared by the preparation method as described above in the preparation of a product for repairing a nerve defect.

[0017] The application has the following beneficial effects: The application provides a conductive composite hydrogel nerve conduit loaded with human endometrial mesenchymal stem cells. The conduit is constructed by crosslinking conductive polypyrrole with natural chitosan and gelatin, to construct a three-dimensional biomimetic scaffold with good biocompatibility, degradability and stable conductivity, successfully simulating the electrophysiological microenvironment required for nerve regeneration. At the same time, the special stem cells loaded can efficiently survive in the conductive microenvironment and exert paracrine function, cooperating with the physical guidance of the material, thereby significantly promoting the directional regeneration of nerve axons, myelination and functional recovery in a large animal model, and having excellent repair effect.

[0018] The product of the application provides a promising alternative for clinical repair of long-segment peripheral nerve defects. The injectable / shapeable property facilitates surgical application, avoids the secondary surgical injury and complications of the donor area of autologous transplantation, and has a clear clinical translation path. The technology is expected to solve the problem of shortage of autologous nerve donors, and provide a safe and effective new tissue engineering repair strategy for patients with peripheral nerve injury, and has important social value and market potential. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a synthesis flowchart (A) and appearance diagram (B) of the PPY-CHI-GEL conductive hydrogel material; Figure 2 It is a scanning electron microscope image (A) of the PPY-CHI-GEL and CHI-GEL hydrogel, a flowchart (B) of in-vitro conductivity test, an electromyogram (C), a statistical diagram of electromyogram (D), and a comparison diagram of conductivity (E); Figure 3 It is Ki67 immunofluorescence staining diagrams (A and B) and fluorescence density statistical diagrams (C) of cell growth and adhesion on the PPY-CHI-GEL composite hydrogel and CHI-GEL hydrogel material; CCK-8 determination of cells on the material after 1 day and 3 days of inoculation (D-E); Figure 4Preparation of animal model of sciatic nerve injury and in vivo experimental flowchart (A); animal model preparation process and sciatic nerve in vitro after nerve conduit access (B); Figure 5 Footprint map collected by walking trajectory analysis of each group after 8 weeks of surgery (A); statistical results of sciatic nerve function index SFI (B); images of gastrocnemius muscle obtained by muscle atrophy recovery analysis (C) (left: muscle tissue after surgery, right: untreated muscle tissue); relative wet weight quantitative analysis of gastrocnemius muscle (D); Masson and H&E staining of gastrocnemius muscle tissue cross section of different groups after implantation for 8 weeks (E); percentage of gastrocnemius muscle area to total area (F); Figure 6 Electrophysiological analysis mode chart for electromyography to collect compound muscle action potential (CMAP) of injured and healthy limbs (A); compound muscle action potential electrophysiological recording of different groups after implantation for 8 weeks (B); amplitude of each group after implantation for 8 weeks (C); Figure 7 Immunofluorescence of nerve cross section (scale = 100 μm). DETAILED DESCRIPTION

[0020] The present application provides a nerve conduit, in the present application, the nerve conduit comprises a three-dimensional conductive composite hydrogel loaded with human endometrial mesenchymal stem cells (EMSCs). The human endometrial mesenchymal stem cells are derived from human endometrial tissue, which is a kind of adult stem cell with multi-directional differentiation potential and strong paracrine function. It can be separated from menstrual blood or endometrial biopsy tissue by standard tissue block adhesion method or enzyme digestion method, and its surface markers (such as high expression of CD73, CD90, CD105, etc.) are identified by flow cytometry. The three-dimensional conductive composite hydrogel is a kind of hydrophilic material with three-dimensional network structure, which can absorb and hold a large amount of water, and has a certain electrical conductivity. Specifically, the composite hydrogel comprises a conductive polymer and a biological material. Among them, "conductive polymer" refers to a kind of high molecular material with conjugated π electron main chain, which can obtain conductivity by doping and other methods; "biological material" refers to a substance derived from nature or synthesis, which can be used for contact and interaction with life system, and can evaluate, treat, enhance or replace any tissue organ function. In the present application, the conductive polymer is grafted on the biological material and crosslinked to form the three-dimensional network. "Grafting" generally refers to connecting one polymer chain to another polymer main chain through chemical bond; "crosslinking" refers to the process of forming chemical bond or physical connection between linear or branched polymer macromolecules, thereby forming a three-dimensional network structure, which can improve the mechanical strength and stability of the material.

[0021] Preferably, the conductive polymer is polypyrrole (PPy). Polypyrrole is a kind of conductive polymer obtained by the oxidative polymerization of pyrrole monomer, which has good environmental stability and biocompatibility, and is often used in the fields of biosensing, neural electrodes and tissue engineering. The biomaterials comprise chitosan (CHI) and gelatin (GEL). Chitosan is a natural linear polysaccharide obtained by deacetylation of chitin, which has good biocompatibility, biodegradability, hemostasis and antibacterial properties, and is a commonly used tissue engineering scaffold material. Gelatin is a product of partial hydrolysis of collagen, which is rich in arginine-glycine-aspartic acid (RGD) sequences, can provide sites for cell adhesion, and has good biological activity and gelation properties.

[0022] The present application also provides a preparation method of the nerve conduit. The method for preparing the nerve conduit can be routinely selected by those skilled in the art according to the material properties and product morphology requirements. The method of the present application includes but is not limited to the following steps: S1. Dissolve chitosan in an acetic acid-containing solution, add pyrrole monomer and oxidizing agent to polymerize pyrrole, and obtain a polypyrrole-chitosan composite solution. The acetic acid-containing solution is used to dissolve chitosan, and acetic acid, as a weak acid, can provide protons to protonate the amino groups on the chitosan molecular chain, thereby dissolving in water. The volume concentration of acetic acid can be 0.5%, 1.0%, 1.5%, or 2.0%, preferably 0.8% to 1.5%, and further preferably about 1.0%. Before adding the oxidizing agent, the mixed system containing chitosan and pyrrole monomer can be treated in the dark for a period of time, for example, 5 to 30 minutes, to avoid premature photooxidation of the pyrrole monomer. The oxidizing agent can be added dropwise to the acetic acid-containing solution at a rate of 0.1 mL / h, 0.5 mL / h, 1.0 mL / h, 1.5 mL / h, or 2.0 mL / h, preferably 0.3 mL / h to 1.2 mL / h, and further preferably about 0.5 mL / h. The dropwise addition helps to control the intensity of the polymerization reaction, allowing the polypyrrole to form more uniformly. The reaction for polymerizing pyrrole is carried out in the dark, and the reaction time can be 6 hours, 12 hours, 24 hours, 48 hours, or 72 hours, preferably 24 to 60 hours, and further preferably about 48 hours. After obtaining the polypyrrole-chitosan composite solution, a step of dialysis purification can also be included to remove small molecular impurities and unreacted monomers. The dialysis purification can use a dialysis bag with a molecular weight cut-off of 8 kDa, 10 kDa, 12 kDa, 14 kDa, or 16 kDa, preferably 10 kDa to 14 kDa. The dialysis purification can be carried out in a phosphate buffer solution (such as 0.1x PBS), and the dialysis solution is replaced 2 to 4 times within 4 to 12 hours, for example, every 4 hours. Before the subsequent cross-linking reaction, a step of adjusting the pH of the polypyrrole-chitosan composite solution to 6.0 to 7.0 is also included, and the pH value can be, for example, 6.0, 6.2, 6.5, 6.8, or 7.0, preferably 6.3 to 6.8, and further preferably about 6.5. The pH adjustment can use an alkali solution commonly used in the art, such as a sodium hydroxide solution.

[0023] S2. Mix the polypyrrole-chitosan composite solution with gelatin in the presence of a cross-linking agent to carry out a cross-linking reaction, forming a polypyrrole-chitosan-gelatin hydrogel. The cross-linking reaction can be initiated by physical mixing, for example, by vortex mixing for 10 seconds, 30 seconds, 60 seconds, 90 seconds, or 120 seconds, preferably 20 seconds to 90 seconds, and further preferably about 30-60 seconds. Mixing promotes the components to come into full contact and initiates the cross-linking process.

[0024] S3. Load human endometrial mesenchymal stem cells into the polypyrrole-chitosan-gelatin hydrogel to obtain the nerve conduit. The method of loading cells can be direct dropwise addition, injection, or blending, etc.

[0025] Preferably, in step S1, the concentration of chitosan in the acetic acid-containing solution is 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0% by weight, preferably 1.0% to 2.5%, and more preferably about 2.0%. In step S1, the mass ratio of the pyrrole monomer to chitosan can be 0.05:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, or 0.5:1, preferably 0.1:1 to 0.4:1, and more preferably about 0.3:1.

[0026] Preferably, in step S1, the oxidant is selected from at least one of ferric chloride, ammonium persulfate, and hydrogen peroxide. These are all commonly used oxidants in the oxidative polymerization of pyrrole. More preferably, the oxidant is ferric chloride (FeCl3), and its molar ratio to the pyrrole monomer can be 0.5:1, 0.8:1, 1.0:1, 1.2:1, 1.5:1, or 2.0:1, preferably 1.0:1 to 1.8:1, and more preferably about 1.5:1.

[0027] Preferably, in step S2, the crosslinking agent is selected from at least one of glutaraldehyde, genipin, and a combination of carbodiimide (such as EDC) and N-hydroxysuccinimide (NHS). Glutaraldehyde is a commonly used bifunctional crosslinking agent; genipin is a natural crosslinking agent with low cytotoxicity; the EDC / NHS combination is often used for the condensation crosslinking of carboxyl and amino groups.

[0028] Preferably, in step S3, the seeding density of human endometrial mesenchymal stem cells can be 0.5 × 10⁻⁶. 6 1.0×10 6 1.5×10 6 Or 2.0×10 6 cells / mL, preferably 0.8 × 10⁻⁶. 6 Up to 1.5×10 6 cells / mL, more preferably about 1.0 × 10⁻⁶. 6 cells / mL. In step S3, after loading the stem cells onto the hydrogel, a co-incubation step is also included to allow the cells to adhere and spread better. The co-incubation time can be 4 hours, 8 hours, 12 hours, 18 hours or 24 hours, preferably 8 to 16 hours, such as overnight incubation.

[0029] This invention also provides the use of the above-described nerve conduit, or the nerve conduit prepared by the above-described method, in the preparation of products for repairing nerve defects. The "nerve defect" refers to an interruption of nerve tissue continuity caused by trauma, disease, or surgery, including but not limited to peripheral nerve defects (such as sciatic nerve, ulnar nerve, and radial nerve defects) and central nervous system injuries. The "product" generally refers to medical devices, medical materials, or pharmaceutical compositions used for the diagnosis, prevention, or treatment of diseases. In this invention, the product is preferably an implantable nerve repair device. This product can be used to bridge nerve ends and provide a supportive microenvironment for nerve regeneration.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example The method for preparing 3D conductive composite hydrogel nerve conduits loaded with hEMSCs provided in this embodiment is as follows: Phase 1: Preparation of PPY-CHI-GEL Conductive Composite Hydrogel Weigh 1g of chitosan powder and slowly add it to a beaker containing 50ml of deionized water and 500μl of glacial acetic acid. The solution is pale yellow. Seal the beaker with sealing film and stir with a magnetic stirrer at 400 rpm until the chitosan is completely dissolved. Slowly add 300 μl of 98% pyrrole monomer solution to the completely dissolved chitosan solution, and continue magnetic stirring for 5 minutes to mix it evenly. At room temperature, wrap the beaker with aluminum foil to protect it from light. Weigh 0.9 g of FeCl3·6H2O and dissolve it in 1 ml of deionized water using a vortex mixer. Using a micro-injection pump, FeCl3 solution was slowly added dropwise to the above mixture at a rate of 0.5 ml / h, while continuously stirring.

[0032] At room temperature, the reaction was carried out in the dark for 48 hours to obtain a black PPY-CHI composite solution; The reaction solution was transferred into a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in 0.1×PBS solution. The dialysate was replaced at 4, 8 and 12 hours to remove small molecule impurities. This is the PPY-CHI conductive composite material solution. Adjust the pH of the above PPY-CHI composite solution to 6.5 using 10 M NaOH; The pH of both the PPY-CHI solution and the pure chitosan (CHI) solution (as a control) was adjusted to the physiological range (approximately 7.4) using a 50% sodium glycerophosphate solution. Take a 15 ml centrifuge tube and add 440 μl of pH-adjusted PPY-CHI solution, 60 μl of 50% sodium glycerophosphate, and 1.2 μl of 4% glutaraldehyde crosslinking agent in sequence. Add pre-cut cylindrical gelatin blocks (e.g., 2 mm in diameter × 10 mm in length) to the mixture. Immediately mix at high speed on a vortex mixer for 30-60 seconds, and you can observe the solution rapidly gelling to form a PPY-CHI-GEL composite hydrogel; The obtained hydrogel scaffold was sterilized by ultraviolet light and kept for later use.

[0033] Phase Two: Isolation, Culture, and Loading of Human Endometrial Mesenchymal Stem Cells (hEMSCs) Primary isolation of endometrial mesenchymal stem cells (hEMSCs) Endometrial tissue from patients with ethical approval and informed consent was physically cut into small fragments and washed three times with PBS. The tissue fragments were placed in centrifuge tubes containing a complex enzyme digestion solution (collagenase III, DNase, 0.05% trypsin, prepared in PBS) and digested at 37°C in a water bath at 70 rpm for 45 minutes. The supernatant was collected and neutralized in DMEM / F-12 complete medium (containing 10% FBS and 1% penicillin antibiotics), and the digestion was repeated three times. The cell suspension was collected, filtered through a 70µm cell sieve, centrifuged, and the supernatant was discarded. The cells were resuspended in fresh medium and seeded into 10 cm culture dishes, incubated at 37°C in a 5% CO2 incubator, with the medium changed every 3 days.

[0034] Passaging and expansion: When the cell confluence reached 90%, the cells were digested with 0.05% trypsin and passaged at a ratio of 1:3. Cells in stable condition from passages 3 to 5 (P3-P5) were used for subsequent experiments.

[0035] Identification: Surface markers of P3 generation cells were detected by flow cytometry. The results showed that the cells highly expressed mesenchymal stem cell markers CD44, CD73, CD90, and CD105 (positive rate >95%), and lowly expressed hematopoietic stem cell markers CD11b, CD34, CD45, and HLA-DR (positive rate <5%), consistent with the immunophenotypic characteristics of mesenchymal stem cells.

[0036] Construction of PPY-CHI-GEL neural conduits loaded with hEMSCs Cell load The prepared and sterilized PPY-CHI-GEL hydrogel scaffolds were placed in sterile culture dishes. hEMSCs cultured to passages P3-P5 were digested with trypsin, counted, and then cultured at a concentration of 1×10⁻⁶. 6The cells were resuspended at a density of cells / mL in a small amount of DMEM / F-12 complete medium. Under aseptic conditions, the cell suspension was slowly added dropwise onto the hydrogel scaffold and incubated overnight at 37°C with 5% CO2 to allow the cells to fully adhere and migrate into the scaffold, thus obtaining the neural conduit loaded with hEMSCs.

[0037] The 3D conductive composite hydrogel nerve conduit loaded with hEMSCs prepared in this embodiment is mainly used to repair peripheral nerve injuries, especially long-segment nerve defects. It can be used as a nerve graft to bridge severed nerve ends during surgery, providing a physical channel, conductive microenvironment, and bioactivity support for nerve regeneration, replacing traditional autologous nerve transplantation or single-function artificial nerve conduits.

[0038] This embodiment provides the product for use as an implantable medical device: Specifications: The hydrogel can be pre-formed or shaped intraoperatively into the required size catheter according to the length of the nerve defect.

[0039] Usage and dosage: In microsurgery, the prepared “PPY-CHI-GEL-EMSCs” nerve conduit is trimmed to a length that matches the nerve defect. The proximal and distal ends of the damaged nerve are inserted into the two ends of the conduit, respectively. The epineurium is then anastomosed and fixed to the conduit wall end-to-end with 9-0 or 10-0 non-traumatic sutures. One conduit of the appropriate size is implanted at each defect site.

[0040] Performance verification experiment: The product was prepared based on the above preparation method. As a control, an equal volume of pure chitosan (CHI) solution was used instead of PPY-CHI solution to prepare CHI-GEL hydrogel according to the same steps.

[0041] Material characterization: Appearance and Microstructure: The macroscopic appearance of PPY-CHI-GEL and CHI-GEL hydrogels was recorded by photography. The prepared hydrogels were dehydrated with gradient concentrations of ethanol (50%, 75%, 90%, 100%), dried, and sputter-coated with gold. The surface microstructure was then observed using scanning electron microscopy (SEM) at ×3000, ×5000, and ×10000 magnification. Results are as follows: Figure 1 and Figure 2 As shown in Figure A. Figure 1 As can be seen, PPY-CHI-GEL appears as uniform black cylinders, while CHI-GEL appears pale yellow. (SEM image) Figure 2 A) shows that PPY-CHI-GEL gel has more pores on its surface and a rough, granular texture, while CHI-GEL gel has fewer pores and a smoother surface overall. This indicates that the porous structure of PPY-CHI-GEL is more conducive to cell adhesion, growth, and substance exchange.

[0042] Electrical conductivity evaluation: (1) Four-probe method: PPY-CHI-GEL and CHI-GEL were prepared into thin films with a thickness of approximately 2 mm. The conductivity was measured at three randomly selected points using a four-probe resistivity meter. The results are as follows: Figure 2 As shown in Figure E, the conductivity of the PPY-CHI-GEL composite hydrogel is (20.6±0.99)×10⁻⁶. - ³ mS / mm, significantly higher than that of CHI-GEL hydrogel (9.925±0.74)×10 - The result of 3 mS / mm (P<0.0001) indicates that the introduction of polypyrrole significantly improves the conductivity of the material.

[0043] (2) Voltage Loss Measurement: To simulate in vitro conductivity, the material was connected to isolated muscle tissue. A positive stimulating electrode was placed at one end of the material, a negative stimulating electrode and a negative receiving electrode were placed at the end of the muscle, and a positive receiving electrode was placed 2 cm away from the end of the muscle. Figure 2 B). A 5 V voltage was applied for stimulation, and the electromyography (EMG) waveform and voltage value were recorded. Results are as follows: Figure 2 As shown in C and 2D, the conduction voltage of the PPY-CHI-GEL group (39.25±12.9 μV) is significantly higher than that of the CHI-GEL group (20.9±3.04 μV), proving that PPY-CHI-GEL has good electrical signal conduction capability.

[0044] In vitro biocompatibility evaluation of PPY-CHI-GEL neural conduits loaded with hEMSCs (1) CCK-8 method: PPY-CHI-GEL and CHI-GEL hydrogels were co-incubated with DMEM / F-12 medium and high-glucose DMEM medium for 24 hours, respectively, and the extracts were collected. hEMSCs and mouse hippocampal neurons (HT22) were seeded at a density of 5000 cells / well in 96-well plates, and 100 μL of the corresponding material extract was added to each well, with ordinary medium as a control. After 1 day and 3 days of culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 1-4 hours. The absorbance (OD value) was measured at 450 nm. The results are as follows: Figure 3 As shown in D-3E, there were no significant differences in OD values ​​among the groups at 1 day and 3 days (P>0.05), indicating that neither hydrogel was cytotoxic.

[0045] (2) Ki67 immunofluorescence staining: hEMSCs and HT22 cells were seeded onto 24-well cell slides containing material extract. After 1 and 3 days of culture, Ki67 immunofluorescence staining was performed, and the cell nuclei were counterstained with DAPI. The cells were observed and photographed under a fluorescence microscope, and the fluorescence density was counted using ImageJ software. The results are as follows: Figure 3 As shown in A-3C, there was no significant difference in the number of Ki67 positive cells and fluorescence density between the experimental group and the control group (P>0.05), further demonstrating that both hydrogels have good cell compatibility and do not affect cell proliferation activity.

[0046] In vivo study of PPY-CHI-GEL nerve conduit loaded with hEMSCs repairing sciatic nerve defects in rats Laboratory animals and grouping Adult male SD rats (8 weeks old, weighing 180-200 g) were randomly divided into 4 groups, with 6 rats in each group: (1) PPY-CHI-GEL-EMSCs group (experimental group): PPY-CHI-GEL neural conduit loaded with hEMSCs was implanted; (2) PPY-CHI-GEL group (material control group): PPY-CHI-GEL nerve conduits without cell loading were implanted; (3) CHI-GEL group (negative control group): CHI-GEL nerve conduit was implanted; (4) Autologous graft group (positive control group): The excised nerve segment was rotated 180° and then anastomosed in situ.

[0047] Establishment and material implantation of a rat model of sciatic nerve defect (1) Anesthesia: Inhalation anesthesia with isoflurane (5% for induction, 2% for maintenance).

[0048] (2) Modeling and Implantation: Rats were fixed in a lateral decubitus position. The skin of the left hind limb was prepared and disinfected. The skin and subcutaneous muscles were incised to expose the left sciatic nerve. A nerve segment of about 10 mm was removed in the middle of the nerve to create a nerve defect. The prepared 10 mm long nerve conduits were bridged to the nerve stump according to the groups. The epineurium and the conduit wall were anastomosed end to end using 9-0 atraumatic sutures. Figure 4 (A, 4B). In the autologous graft group, the excised nerve segments were directly anastomosed in situ.

[0049] (3) Postoperative management: The muscles and skin were sutured in layers and disinfected with povidone-iodine. Penicillin was injected intraperitoneally for 3 consecutive days after surgery to prevent infection. Functional and histological examinations were performed on rats in each group 8 weeks after surgery.

[0050] Functional recovery evaluation (1) Sciatic Nerve Function Index (SFI): Eight weeks post-surgery, rats were dipped in red ink on their hind limbs and allowed to walk through a narrow passage lined with white paper to collect footprints. Footprint length (PL), toe width (TS), and middle toe width (IT) were measured on the normal side (N) and experimental side (E). SFI was calculated using the following formula: SFI = [109.5×(ETS - NTS) / NTS - 38.3×(EPL - NPL) / NPL - 13.3×(EIT -NIT) / NIT - 8.8]. The closer the SFI value is to 0, the better the function; -100 indicates complete loss of function.

[0051] The results are as follows Figure 5 As shown in A-5B, the SFI value of the PPY-CHI-GEL-EMSCs group (-54.15±4.16) was significantly higher than that of the PPY-CHI-GEL group (-66.18±2.85) and the CHI-GEL group (-83.20±3.64), and there was no statistical difference compared with the autologous graft group (-53.20±4.53). This indicates that the conductive catheter loaded with stem cells can most effectively promote the recovery of sciatic nerve motor function.

[0052] (2) Electrophysiological assessment: Eight weeks post-surgery, rats were re-anesthetized to expose the sciatic nerve. The amplitude of the compound muscle action potential (CMAP) of the gastrocnemius muscle was recorded using an electromyography (EMG) instrument. The stimulating electrode was placed proximal to the nerve, and the recording electrode was inserted into the gastrocnemius muscle. Results are as follows: Figure 6 As shown in A-6C, the CMAP amplitude recovery rate of the PPY-CHI-GEL-EMSCs group (67.28±4.99%) was significantly higher than that of the PPY-CHI-GEL group (35.70±12.46%) and the CHI-GEL group (7.8±6.31%), and comparable to that of the autologous graft group (69.87±1.63%), indicating that it can better reconstruct neural conduction pathways.

[0053] (3) Gastrocnemius muscle atrophy analysis: After sacrificing the rats, the bilateral gastrocnemius muscles were separated and weighed wet. The wet weight ratio of the gastrocnemius muscle (weight of the operated side / weight of the normal side) was calculated. At the same time, a portion of the muscle tissue was taken for HE and Masson staining to observe the morphology of the muscle fibers, and the cross-sectional area of ​​the muscle fibers was counted using ImageJ software.

[0054] The results are as follows Figure 5 As shown in C-5F, the wet weight ratio of the gastrocnemius muscle in the PPY-CHI-GEL-EMSCs group (64.57±1.85%) was significantly higher than that in the two material control groups, and close to that in the autologous graft group (69.87±1.63%). Histological staining showed that the muscle fibers in this group were more regularly arranged, had the least degree of atrophy, and had the largest cross-sectional area of ​​muscle fibers, indicating the best recovery of nerve function and effective reinnervation of the target organ.

[0055] Histological evaluation of nerve regeneration Eight weeks post-surgery, nerve tissue from the bridging site was harvested, fixed, embedded, and sectioned.

[0056] (1) HE staining: Observe the overall morphology of nerve regeneration. Results are as follows: Figure 5 As shown in E, tissue proliferation was observed in each group of nerves, and the material was well integrated with the nerve tissue.

[0057] (2) Immunofluorescence staining: The nerve cross sections were subjected to double immunofluorescence staining with S-100 (Schwann cell marker) and NF-200 (axon marker), and the cell nuclei were counterstained with DAPI. The cells were observed and photographed under a fluorescence microscope.

[0058] The results are as follows Figure 7 As shown, the S-100 (green) and NF-200 (red) positive areas in the PPY-CHI-GEL-EMSCs group and the autologous graft group were the largest and had the highest fluorescence intensity. Moreover, the two were well co-localized, indicating that a large number of mature myelin sheaths and axons were formed in the regenerated nerves in this group. Their histological morphology was closest to that of normal nerves, which was significantly better than that of the PPY-CHI-GEL group and the CHI-GEL group.

[0059] As demonstrated by the above embodiments, this invention provides a conductive composite hydrogel nerve conduit loaded with human endometrial mesenchymal stem cells. In vitro experiments have shown that this conduit material possesses suitable conductivity, a porous microstructure, and good cell compatibility, supporting stem cell survival and growth. In vivo experiments in a rat sciatic nerve defect model further demonstrate that implantation of this nerve conduit effectively promotes morphological regeneration and functional recovery of the injured nerve. In several key evaluation indicators, including sciatic nerve function index, muscle action potential recovery, reduction of target muscle atrophy, and myelin axon regeneration, its repair effect is significantly better than the control group without stem cells or without conductivity, and comparable to the repair level of the autologous nerve transplantation group. Therefore, this invention provides an effective tissue engineering repair strategy for peripheral nerve injuries.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nerve conduit, characterized in that, The invention includes a three-dimensional conductive composite hydrogel loaded with human endometrial mesenchymal stem cells, wherein the composite hydrogel comprises a conductive polymer and a biomaterial, and the conductive polymer is grafted onto the biomaterial and cross-linked thereto to form a three-dimensional network.

2. The nerve conduit according to claim 1, characterized in that, The conductive polymer is polypyrrole, and the biomaterial comprises chitosan and gelatin.

3. The method for preparing the nerve conduit according to claim 1 or 2, characterized in that, Includes the following steps: S1. Chitosan is dissolved in a solution containing acetic acid, and pyrrole monomer and oxidant are added to polymerize pyrrole to obtain a polypyrrole-chitosan complex solution; S2. The polypyrrole-chitosan complex solution is mixed with gelatin in the presence of a crosslinking agent to carry out a crosslinking reaction, forming a polypyrrole-chitosan-gelatin hydrogel; S3. Human endometrial mesenchymal stem cells are loaded into the polypyrrole-chitosan-gelatin hydrogel to obtain the neural conduit.

4. The preparation method according to claim 3, characterized in that, In step S1, the volume concentration of acetic acid in the acetic acid-containing solution is 0.5% to 2%; In step S1, the mixture containing chitosan and pyrrole monomers is protected from light before the oxidant is added. In step S1, the oxidant is added dropwise to the acetic acid-containing solution at a rate of 0.1 mL / h to 2 mL / h; In step S1, the pyrrole polymerization reaction is carried out under light-protected conditions for 6 to 72 hours; In step S1, after obtaining the polypyrrole-chitosan complex solution, a dialysis purification step is also included. The dialysis purification is carried out using a dialysis bag with a molecular weight cutoff of 8 kDa to 16 kDa, in phosphate buffer, and the dialysis solution is changed 2 to 4 times within 4 to 12 hours. In step S1, prior to the crosslinking reaction, the pH of the polypyrrole-chitosan complex solution is adjusted to 6.0 to 7.

0.

5. The preparation method according to claim 3, characterized in that, In step S2, the crosslinking reaction is initiated by vortex oscillation mixing for 10 to 120 seconds.

6. The preparation method according to claim 3, characterized in that, In step S1, the concentration of chitosan in the acetic acid-containing solution is 0.5% to 3% by weight. In step S1, the mass ratio of the pyrrole monomer to chitosan is 0.05:1 to 0.5:

1.

7. The preparation method according to claim 3, characterized in that, In step S1, the oxidant is selected from at least one of ferric chloride, ammonium persulfate, and hydrogen peroxide.

8. The preparation method according to claim 7, characterized in that, The oxidant is ferric chloride, and the molar ratio of ferric chloride to pyrrole monomer is 0.5:1 to 2:

1.

9. The preparation method according to claim 3, characterized in that, In step S2, the crosslinking agent is selected from at least one of glutaraldehyde, genipin, and a combination of carbodiimide and N-hydroxysuccinimide; In step S3, the seeding density of human endometrial mesenchymal stem cells used is 0.5×10^6 to 2×10^6 cells / mL; In step S3, after loading the stem cells onto the hydrogel, a co-incubation step of 4 to 24 hours is also included.

10. Use of the nerve conduit according to claim 1 or 2, or the nerve conduit prepared by any one of claims 3 to 9, in the preparation of products for repairing nerve defects.

Images