Preparation and application of an electrospun nanofiber double-layer nerve conduit

Abstract

The invention discloses a preparation and application of an electrospun nanofiber double-layer nerve conduit, which relates to the technical field of medical supplies. The double-layer nerve conduit is APML@PC, which is composed of PVA-CMCS and PCL-MoS2-LN electrospinning membranes. APML@PC is set as a conduit structure, the inner layer of the conduit of APML@PC is a PCL-MoS2-LN electrospinning membrane, and the outer layer of the conduit of APML@PC is PVA-CMCS. APML@PC is used for the preparation and treatment of peripheral nerve injury. Through the ordered topological structure and the simulation of the microenvironment of the neural tissue in the body, the topographic clues and biological clues are transmitted to promote the adhesion and proliferation of stem cells and induce their differentiation into Schwann-like cells, thereby promoting myelination and neurite regeneration through the continuous secretion of neurotrophic factors.

Description

Technical Field

[0001] The invention relates to the technical field of medical supplies, and in particular to the preparation and application of an electrostatically spun nanofiber double-layer nerve conduit. Background Art

[0002] Peripheral nerve injury is a common clinical disease, mainly caused by metabolic disorders, trauma, infection and other factors, which seriously reduces people's quality of life and brings huge medical and economic burden to society. Unlike the central nervous system, neurons in the peripheral nervous system have a certain degree of regeneration and growth ability after injury, but the slow rate and inaccurate direction of axon extension hinder the repair of peripheral nerves. Autologous nerve transplantation is still the preferred method for repairing long gaps in peripheral nerves. However, factors such as insufficient donor nerves, functional limitations of donor sites, mismatched nerve fibers, postoperative nerve twisting or dislocation, neuromas and immunosuppression have severely limited its clinical application. Nerve conduits are an alternative to autologous nerve transplantation, which can connect the proximal and distal ends of the nerve defect and provide physical and biochemical guidance for axon regeneration.

[0003] Schwann cells (SCs) play a vital role in the regeneration of peripheral nerves. After nerve injury, SCs first recruit macrophages to clear myelin debris, then proliferate and arrange longitudinally to form Büngner bands, providing longitudinal guidance channels for axon regeneration at the nerve stump. In addition, SCs can secrete neurotrophic factors to support axon growth and myelination, thereby further promoting nerve regeneration. Unfortunately, in most experimental and clinical studies, the source of donor SCs is limited by the low proliferation capacity of adult peripheral nerve cells and the high morbidity of donor sites. Interestingly, multiple types of mesenchymal stem cells can be induced into SCs-like cells, such as adipose-derived mesenchymal stem cells (ADSCs), bone marrow-derived mesenchymal stem cells (BMSCs), umbilical cord-derived mesenchymal stem cells (UCSCs), and dental pulp-derived mesenchymal stem cells (DPSCs). In particular, ADSCs have become promising seed cells for the treatment of peripheral nerve injury and repair because of their huge differentiation potential and ease of acquisition through autologous, minimally invasive methods. However, there are still problems such as low cell survival rate and incomplete differentiation during ADSC transplantation, which limits its effect in nerve repair.

[0004] Highly oriented topological structures can mimic the ordered structure of neural tissue, guide the orderly arrangement of SCs, and promote the differentiation of stem cells into nerves and the directional extension of neurites, showing great potential in promoting the repair of peripheral nerve injuries. The diameter of electrospun nanofibers can be controlled in the range of 50-500 nm and can be easily collected into an ordered topological structure to mimic the size of collagen fibers in the natural extracellular matrix (ECM) and the orientation of ECM in neural tissue. In particular, PCL fibers, because of their microstructure similar to the size of peripheral nerve ECM, have biocompatibility and excellent mechanical properties. However, the hydrophobic surface of PCL is not conducive to the adhesion of nerve cells, affecting the longitudinal guidance function of regenerated axons. To solve this problem, the surface of PCL fiber scaffolds can be hierarchically modified with ECM bioactive factors (laminin, LN) to enhance biochemical guidance, further affect the differentiation of stem cells and manipulate the migration of stem cells.

[0005] In recent years, scaffolds based on hybrid inorganic two-dimensional nanomaterials have shown great potential in controlling stem cell differentiation by providing controllable physical, chemical, and biological properties to regulate cell-matrix interactions. 2 ) because it is an unsaturated d orbital of MX 2 The compound has excellent physical and chemical properties, good conductivity, and biocompatibility, and has attracted extensive attention in the biomedical field. It is worth noting that bulk three-dimensional crystalline MoS 2 The stacking structure has strong covalent bonds between Mo and S and MoS 2 Weak van der Waals interactions between adjacent layers allow the preparation of single-layer or few-layer MoS by top-down approaches 2 This layered MoS 2 It has an "S-Mo-S" sandwich structure, ultrathin direct band gap, high surface area and specific functional groups, which are beneficial to regulating the assembly of ECM proteins and stem cell adhesion and growth. However, it has not been used in the preparation of double-layer nerve conduits.

[0006] In addition to the microstructure of the internal filler, the mechanical properties and porosity of the tubular sheath are equally important for the nerve repair effect, which is often overlooked in the previous scaffold design. Among the many nerve tissue engineering materials, the hydrogel prepared by the general physical freeze-thaw method of polyvinyl alcohol (PVA) has a good network structure and biocompatibility, and can be modified by adding functional materials to show excellent mechanical properties and degradability. Chitosan, as a natural polysaccharide, has good compatibility with nerve cells and can promote the attachment, differentiation and growth of nerve cells. However, the chitosan used as a scaffold so far is insoluble in water and has a slow and uncontrollable decomposition rate. The degradation rate does not match the regeneration rate of new tissue, which limits the application of chitosan in nerve repair. The introduction of active hydroxyl groups at the C6 position of chitosan to obtain carboxymethylated chitosan (CMCS), which has a structure similar to the polysaccharide functional factors in natural ECM, has better water solubility, stronger biocompatibility, degradability and moisturizing ability than chitosan, and can promote the proliferation of SCs. Therefore, it has broad prospects in the field of medical nerve materials. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation and application of an electrospun nanofiber double-layer nerve conduit, wherein a soft and porous polyvinyl alcohol-carboxymethyl chitosan (PVA-CMCS) is used as the outer layer of the double-layer nerve conduit, and an ordered polycaprolactone-molybdenum disulfide cross-linked laminin (PCL-MoS 2 -LN) electrospun membrane is rolled into polyvinyl alcohol-carboxymethyl chitosan; PVA-CMCS is abbreviated as PC, PCL-MoS 2 -LN is abbreviated as APML; thus constructing PCL-MoS 2 -LN@PVA-CMCS double-layer nerve conduit, PCL-MoS 2 -LN@PVA-CMCS is abbreviated as APML@PC. The constructed double-layer nerve conduit transmits biological cues and topological cues, aiming to promote the adhesion, proliferation and differentiation of stem cells into SCs in an efficient way.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] An electrospun nanofiber double-layer nerve conduit, the double-layer nerve conduit is APML@PC, and APML@PC is composed of PVA-CMCS and PCL-MoS 2 -LN electrospun membrane forms a double-layer nerve conduit;

[0010] APML@PC is set as a conduit structure, and the inner layer of the conduit of APML@PC is PCL-MoS 2-LN electrospun membrane, the outer layer of the APML@PC conduit is PVA-CMCS;

[0011] Application of APML@PC as a nerve conduit for the treatment of peripheral nerve injury.

[0012] Further: The preparation method of the electrospun nanofiber double-layer nerve conduit comprises the following steps:

[0013] Step 1) Inner layer preparation: MoS 2 Nanosheet powder is added to HFIP solution and ultrasonically obtained into dispersion liquid, and then PCL is added into the dispersion liquid and stirred to obtain spinning solution;

[0014] The spinning solution was prepared into ordered fiber membrane PCL-MoS in an electrospinning machine. 2 , ordered fibrous membrane PCL-MoS 2 MES / EDC / NHS solution was added, incubated at room temperature, and then immersed in LN and shaken to obtain an ordered fiber membrane of cross-linked LN, which is PCL-MoS 2 -LN electrospun membrane;

[0015] Step 2) Preparation of outer layer: PVA and deionized water are mixed and heated and stirred to obtain a PVA solution, and then CMCS is added and stirred. After stirring, the solution is injected into a mold, and a stainless steel stick is inserted in the middle to seal the two ends of the mold. The solution is frozen and thawed, and demolded to obtain a nerve conduit PVA-CMCS.

[0016] Step 3) Preparation of double-layer nerve conduit: PCL-MoS 2 The -LN electrospun membrane was cut into rectangles, rolled up with a stainless steel stick and inserted into the PVA-CMCS nerve conduit to prepare a double-layer nerve conduit APML@PC.

[0017] Further: In step 1), the concentration of the LN solution is 0.2-2 mg / mL.

[0018] Further: in the step 2), the mold is selected as a polyethylene tetrafluoro tube mold, the length of the polyethylene tetrafluoro tube mold is 0.5-5 cm, and the inner diameter is 1-3 mm.

[0019] Further: in the step 2), a stainless steel stick with a diameter of 0.5-2.5 mm is inserted in the middle.

[0020] Further: In step 3), PCL-MoS 2 -LN electrospun membranes were cut into rectangles with a length of 6-24 mm and a width of 2.5-10 mm.

[0021] Further: in the step 3), a stainless steel stick with a diameter of 0.5-2.5 mm is used to roll up and insert into the single-layer nerve conduit.

[0022] Further: in the step 2), the freezing and thawing cycles are 4-8 times.

[0023] Further: the APML@PC was immersed in PBS and then shaken, taken out after 2-12 weeks, freeze-dried and weighed, and the in vitro weight loss rate was calculated.

[0024] Further: the APML@PC is cut and sterilized, then added to a physiological saline warm water bath for incubation and set aside;

[0025] Fresh blood of rats was anticoagulated with sodium citrate and centrifuged to separate red blood cells. After repeated washing with physiological saline, a red blood cell suspension was prepared. The cell suspension was aspirated and incubated in the reserved APML@PC. After incubation, physiological saline was added to stop hemolysis. The incubation was continued at a constant temperature. After the incubation, the blood was centrifuged and the supernatant was taken to detect the amount of hemoglobin released and the hemolysis rate was calculated.

[0026] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0027] (1) An electrospun nanofiber double-layer nerve conduit with an inner layer made by electrospinning orderly arranged PCL-MoS 2 -LN electrospun membrane, the outer layer is prepared by physical freeze-thaw to prepare PVA-CMCS nerve conduit; simulate the nerve fiber bundle structure of natural nerve tissue, promote the adhesion, proliferation and neural differentiation of stem cells.

[0028] (2) The outer layer is a soft and porous polyvinyl alcohol-carboxymethyl chitosan (PVA-CMCS), which can promote the exchange of oxygen and nutrients while preventing the infiltration of fibrous tissue; the inner layer is an ordered polycaprolactone-molybdenum disulfide cross-linked laminin (PCL-MoS 2 -LN) electrospun membranes can transmit topographical and biological cues through ordered topological structures and simulated neural tissue microenvironment in vivo to promote stem cell adhesion and proliferation and induce their differentiation into Schwann-like cells, thereby promoting myelination and neurite regeneration through continuous secretion of neurotrophic factors.

[0029] (3) The double-layer nerve guide of the present invention has suitable degradability and good blood compatibility.

[0030] (4) The double-layer nerve conduit of the present invention was successfully implanted into the 10 mm defect of the sciatic nerve of SD rats, which can accelerate axonal growth, promote myelin regeneration and improve nerve function recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a scanning electron microscope image of the electrospun nanofiber double-layer nerve conduit prepared by the present invention.

[0032] Figure 2 This is the in vitro degradation diagram of the electrospun nanofiber double-layer nerve conduit in Example 2.

[0033] Figure 3 This is a diagram of the in vitro hemolysis experiment of the electrospun nanofiber double-layer nerve conduit in Example 3.

[0034] Figure 4 In Example 4: Figure A shows different groups of nerve conduits implanted in 10 mm sciatic nerve defects in rats; Figure B shows representative images of the footprints of the normal side (left) and experimental side (right) of each group of rats when walking; Figure C shows the compound motor action potential waveforms of the distal (upper) and proximal (lower) ends of the stimulation defect on the experimental side of each group of rats.

[0035] Figure 5 In Example 5: Figure A is a morphological observation diagram of the gastrocnemius muscle, wherein the left side is the experimental side and the right side is the normal side, scale bar: 1 cm; Figure B is a Masson's trichrome staining image of the gastrocnemius muscle of the experimental group, scale bar: 100 μm.

[0036] Figure 6 Immunofluorescence image of the cross section of the middle segment of the regenerated nerve in Example 6, blue: DAPI; red: MBP; green: NF200; scale bars are 100 μm and 20 μm, respectively.

[0037] Figure 7 This is the morphological evaluation of axon remyelination in each group using TB and TEM 12 weeks after surgery in Example 7. Toluidine blue staining shows the myelin sheath of regenerated axons. Scale bar: 20 μm.

[0038] Figure 8 The figure is a flow chart of the preparation of the electrospun nanofiber double-layer nerve conduit prepared by the present invention.

[0039] Fig. 9 This is a statistical diagram of the sciatic nerve function index (SFI) in Example 4.

[0040] Fig.10 This is a statistical diagram of compound motor action potentials in Example 4.

[0041] Fig.11 It is a statistical diagram of nerve conduction velocity in Example 4.

[0042] Fig.12 This is a weight ratio diagram of the gastrocnemius muscle in Example 5.

[0043] Fig.13 This is a statistical diagram of the cross-sectional area of ​​the gastrocnemius muscle fibers in the experimental group in Example 5.

[0044] Fig.14 This is a statistical analysis of the myelin thickness of each group in Example 6.

[0045] Fig.15 These are representative TEM images of myelinated axon regeneration 12 weeks after surgery in Example 7. The scale bars are 10 μm at low power, 2 μm at medium power, and 1 μm at high power.

[0046] Fig.16 This is a statistical graph of the axon diameter of each group quantitatively analyzed in Example 7.

[0047] Fig.17 This is a statistical graph of the ratio of the medial axon area to the lateral myelin sheath area of ​​each group quantitatively analyzed in Example 7. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0049] An electrospun nanofiber double-layer nerve conduit, the double-layer nerve conduit is APML@PC, APML@PC has an outer layer of PVA-CMCS and an inner layer of PCL-MoS 2 -Double-layer nerve conduit made of LN electrospun membrane.

[0050] A method for preparing an electrospun nanofiber double-layer nerve conduit comprises the following steps:

[0051] Step 1) Inner layer preparation: MoS 2 Nanosheet powder is added to HFIP solution and ultrasonically obtained into dispersion liquid, and then PCL is added into the dispersion liquid and stirred to obtain spinning solution;

[0052] The spinning solution was prepared into ordered fiber membrane PCL-MoS in an electrospinning machine. 2 , ordered fibrous membrane PCL-MoS 2 MES / EDC / NHS solution was added, incubated at room temperature, and then immersed in LN and shaken to obtain an ordered fiber membrane of cross-linked LN, which is PCL-MoS 2 -LN electrospun membrane;

[0053] Step 2) Preparation of outer layer: PVA and deionized water are mixed and heated and stirred to obtain a PVA solution, and then CMCS is added and stirred. After stirring, the solution is injected into a mold, and a stainless steel stick is inserted in the middle to seal the two ends of the mold. The solution is frozen and thawed, and demolded to obtain a nerve conduit PVA-CMCS.

[0054] Step 3) Preparation of double-layer nerve conduit: PCL-MoS 2The -LN electrospun membrane was cut into rectangles, rolled up with a stainless steel stick and inserted into the PVA-CMCS nerve conduit to prepare a double-layer nerve conduit APML@PC.

[0055] In the following examples, PCL (polycaprolactone), MoS 2 Molybdenum disulfide, HFIP, MES, EDC, NHS, laminin, and CMCS were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China); PVA (polyvinyl alcohol) 1799 was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China); S100β, goat anti-mouse Alexa Fluor® 488, and goat anti-rabbit Alexa Fluor® 594 antibodies were purchased from Abcam (UK); NF200 was purchased from CST (USA); GFAP antibody was purchased from Sanying Biotechnology Co., Ltd. (Wuhan, China); and modified Masson’s trichrome staining kit was purchased from Solebo Technology Co., Ltd. (Beijing, China). Deionized water used in the experiment came from Milli-Q Gradient A10 water purification system.

[0056] Example 1

[0057] 0.1 g MoS was accurately weighed using an electronic analytical balance 2 The nanosheet powder was added to 10 mL of HFIP solution and ultrasonicated for 30 min to fully disperse it. Then 1.2 g of PCL particles were mixed into the MoS 2 The nanosheet dispersion was stirred at room temperature overnight to obtain a spinning solution, and the spinning solution was used to prepare ordered nanofibers using an electrospinning machine, that is, to prepare ordered fiber membrane PCL-MoS 2 The parameters of the electrospinning machine are: flow rate: 1 mL / h, voltage 10 kV, needle tip distance 15 cm to the receiving plate, speed 2800 rpm / min, spinning duration 2 h. 2 MES / EDC / NHS solution was added, the concentration of MES solution in the MES / EDC / NHS solution was 0.1 mol / L, the concentration of NHS was 6 mg / mL, and the concentration of EDC was 4 mg / mL. The mixture was incubated at room temperature for 1 h, and then immersed in laminin and slightly shaken overnight at 4 °C. The ordered fiber membrane of cross-linked LN was obtained, which was PCL-MoS 2 -LN electrospun membrane; ready for use.

[0058] Weigh 3 g of PVA (polyvinyl alcohol) in a single-necked flask, add 30 mL of deionized water, heat and stir in a 95 °C oil bath for 2 h to dissolve it completely, then turn off the heating button and continue stirring overnight. Add 0.9 g of CMCS (carboxymethyl chitosan) to the PVA solution and stir evenly at room temperature to obtain a PVA-CMCS (outer layer is polyvinyl alcohol-carboxymethyl chitosan) solution. Inject the PVA-CMCS solution into a polyethylene tetrafluoroethylene tube mold, insert a stainless steel stick in the middle, and seal both ends of the mold. After freezing and reheating cycles, demold the nerve conduit PVA-CMCS.

[0059] PCL-MoS 2 -LN electrospun membrane was cut into long rectangles and rolled up with a stainless steel rod and inserted into PVA-CMCS, where the ordered direction was consistent with the axial direction of the stainless steel rod, and the direction of the ordered arrangement of the nanofibers was the same as the length direction of the stainless steel rod, thus preparing a double-layer nerve conduit.

[0060] The specific parameter settings in each test case during the preparation of the double-layer nerve conduit APML@PC are shown in Table 1.

[0061] Table 1 Parameters of various experimental examples for preparing double-layer nerve conduits APML@PC

[0062]

[0063] The prepared double-layer nerve conduit APML@PC was observed by SEM. Figure 1 As shown, the inner layer is PCL-MoS 2 -LN electrospinning membrane provides a microenvironment for the adhesion, proliferation, migration and differentiation of stem cells, while the outer layer is a loose and porous PVA-CMCS, which is conducive to the removal of myelin and axons of damaged nerves. This catheter has mechanical properties similar to those of nerve tissue and good biocompatibility.

[0064] Example 2

[0065] In Example 1, PC and APL@PC (APL@PC is a double-layer nerve conduit, and APL is prepared with less MoS than APML) 2 The preparation process was carried out without adding MoS 2 The preparation method is consistent with that of Example 1, except that MoS 2The obtained PCL-LN was inserted into PVA-CMCS and the APML@PC nerve conduit was immersed in PBS (phosphate buffered saline) at 37 °C and shaken at 100 rpm / min. The samples were taken out at 2, 4, 6, 8, 10, and 12 weeks, washed with water to remove excess salt, and weighed after freeze-drying. The in vitro weight loss rate (%) was calculated to estimate the degree of weight change in each time interval.

[0066] Weight loss rate (%) =

[0067] Where: Wi is the mass of the dry sample before immersion; Wx is the mass of the dry sample after immersion.

[0068] like Figure 2 As shown in the figure, the mass loss of each group of samples exceeded 40% at 2 weeks, which was mainly due to the rapid degradation of the conduit due to the water solubility of CMCS. However, by 12 weeks, the mass loss of the PC group (PVA-CMCS single-layer nerve conduit), APL@PC group and APML@PC group was 84.47%, 69.25% and 78.12%, respectively, proving that the conduit not only has a certain degree of degradability, but also can maintain the structure of the conduit, thereby ensuring stability in the process of supporting nerve repair in vivo.

[0069] Example 3

[0070] The PC, APL@PC, and APML@PC nerve conduits were cut into 5 mm in length, sterilized, transferred to 1.5 mL EP (plastic) centrifuge tubes, added with physiological saline, and incubated in a 37 °C constant temperature water bath overnight.

[0071] Fresh blood from male SD rats (200-220 g) was collected and placed in a sodium citrate anticoagulant tube under vacuum and centrifuged at 3000 rpm / min for 15 min to separate red blood cells. An appropriate amount of saline was added to rinse repeatedly, and a 2% w / v red blood cell suspension was prepared, and 200 μL was pipetted into each nerve conduit group. After incubation for 10 min, 1 mL of saline was added to stop hemolysis, and the cells were incubated in a 37 ℃ constant temperature water bath for 1 h. 1 mL of saline was used as the negative control group and 1 mL of 1% TritonX-100 (TritonX-100 is a cell permeabilizing agent that ruptures red blood cells) was used as the positive control group, and the cells were incubated in a 37 ℃ constant temperature water bath for 1 h. At the end of incubation, the cells were centrifuged at 3000 rpm / min for 20 min in a 4°C low-temperature centrifuge. 50 μL of the supernatant was added to a 96-well plate. The amount of hemoglobin released was detected at 540 nm on an ELISA reader, and the hemolysis rate (%) was calculated.

[0072] Hemolysis rate (%) =

[0073] Where: A s is the sample absorbance value; A nc is the absorbance value of negative control; A pc is the absorbance value of the positive control.

[0074] like Figure 3 As shown, 1% Triton X-100 was used as the positive control and saline was used as the negative control. Compared with the positive control group, the hemolysis rates of PC, APL@PC, and APML@PC groups were all less than 2%, indicating that these catheter materials have good blood compatibility.

[0075] Example 4

[0076] 1.0×10 4 ADSCs (adipose-derived mesenchymal stem cells) were seeded on the nanofiber membrane and induced with neural induction medium. After 14 days, the membrane was rolled up and inserted into a PVA-CMCS catheter with an inner diameter of 1.25 mm, an outer diameter of 2.0 mm, and a length of 12 mm. The establishment of a nerve catheter bridging a 10 mm sciatic nerve defect model in SD rats. The experiment was divided into four groups: PC group, APL@PC group, APML@PC group, and autologous nerve transplantation group (Autograft group).

[0077] Sixty SD male rats were randomly divided into four groups (15 rats in each group). They were fasted for 12 h before surgery and anesthetized with 3% pentobarbital solution (30 mg / kg) by intraperitoneal injection. After depilation of the right hind limb, iodine disinfection, sterilized surgical drapes were laid, and observation was performed under a stereoscope. An 8 mm section of the sciatic nerve in the middle of the thigh (near the obturator tendon) was removed, leaving a 10 mm gap after the nerve was retracted. The corresponding nerve conduit was bridged with 11 / 0 absorbable nylon sutures for suture. In the autologous nerve transplantation group, the nerve was cut and reversed 180°, and finally the muscles and skin of the rats were sutured layer by layer with 5 / 0 absorbable sutures. Seven days after surgery, penicillin was injected into the rats' muscles to avoid infection to ensure the smooth progress of the experiment.

[0078] Gait analysis was performed at 12 weeks after nerve conduit transplantation to evaluate the motor recovery of rats in each group. A walking platform was set up, and the rats were starved for 12 h before the test. The rats walked from one end of the walking platform to the other end. A high-speed camera was used to record the walking trajectory of each rat as it passed through the runway. The VisuGait small animal gait analysis system was used to measure and calculate the sciatic nerve function index (SFI) according to the formula.

[0079] Sciatic Function Index (SFI) = + + - 8.8

[0080] Wherein, N: normal or uninjured side (left leg); E: experimental or injured side (right leg); PL: length of rat footprint; IT: distance between the 2nd and 4th toes; TS: distance between the 1st and 5th toes. The SFI range is 0 ~ -100, with higher values ​​indicating better sciatic nerve function and lower values ​​indicating worse function. Each rat was evaluated at least 3 times.

[0081] After motor function assessment, SD rats were anesthetized with pentobarbital (30 mg / kg) and the sciatic nerve was exposed for electrophysiological testing. The recording electrode was inserted into the belly of the gastrocnemius muscle, and the ground electrode was inserted into the tail. The distal and proximal sciatic nerve trunks of the nerve guide were electrically stimulated (3 mA, 1 Hz) in turn, and the compound muscle action potential and nerve conduction velocity were collected and recorded. Each group of experiments was repeated 6 times.

[0082] like Figure 4 As shown in A, to verify the effect of APML@PC double-layer nerve conduits on peripheral nerve regeneration in vivo, PC, APL@PC and APML@PC nerve conduits were implanted into 10 mm sciatic nerve defects in SD rats. Figure 4 As shown in B, 12 weeks after surgery, gait analysis images were first recorded and the sciatic nerve function index (SFI) was calculated to evaluate the functional recovery of peripheral nerve defects in vivo. A more negative SFI value indicates more severe functional impairment, while an SFI value close to zero indicates normal functional recovery. The SFI of rats in the Autograft group and APML@PC group were better than those in the PC group and APL@PC group.

[0083] like Fig. 9 The results showed that the SFI of rats in the Autograft group and APML@PC group were better than those in the PC group and APL@PC group. Peripheral nerves, as a bridge connecting the central nervous system and sensory / motor terminals, can achieve bidirectional transmission of bioelectric signals.

[0084] like Figure 4 C. Fig.10 , Fig.11As shown in Figure 4, in order to further evaluate the reinnervation of the gastrocnemius muscle distal to the injured nerve, the rats were subjected to electrophysiological tests of compound motor action potential and nerve conduction velocity. The enhancement of the peak value of compound motor action potential indicates an increase in the density of regenerated nerve fibers and an improvement in nerve function. Representative compound motor action potential and nerve conduction velocity of the four groups are shown in Figure 4 EF. Among them, except for the Autograft group, the peak value of compound motor action potential of APML@PC (29.91±5.73 mV) was higher than that of PC group (9.85±2.61 mV) and APL@PC group (23.97±4.46 mV). In addition, the nerve conduction velocity of APML@PC group (47.9±4.56 m / s) was also significantly higher than that of PC group (14.8±3.77 m / s) and APL@PC group (28.73±5.71 m / s), which was close to that of Autograft (48.3±3.74 mV). This indicates that APML@PC can increase the action potential amplitude and accelerate nerve conduction velocity, thereby promoting nerve function recovery.

[0085] Example 5

[0086] Twelve weeks after surgery, the gastrocnemius muscles of the experimental side (GM(E)) and the normal side (GM(N)) were weighed, and the atrophy of the gastrocnemius muscles of rats in each group was compared. The gastrocnemius muscle wet weight ratio (GM) was calculated according to the following formula.

[0087] Gastrocnemius muscle weight ratio (%) =

[0088] The middle gastrocnemius muscle was immersed in 4% paraformaldehyde for 48 h, dehydrated with gradient alcohol (50%, 70%, 80%, 90%, 95%, 100%) for 30 min, transparentized with xylene for 5 min, immersed in wax at 60 °C for 3 h, and then embedded in paraffin to make 4 μm pathological sections. Masson's trichrome staining was performed according to the reagent manufacturer's instructions, and the sections were sealed with neutral resin and observed under an optical microscope. At least 5 random fields were selected for each section. Image J software was used to quantify the cross-sectional area of ​​muscle fibers in each group.

[0089] like Figure 5 As shown in A, the bilateral gastrocnemius muscles of rats in each group were separated, photographed and weighed. The digital photos of the gastrocnemius muscles showed that the right side was a healthy gastrocnemius muscle, and the left side was a gastrocnemius muscle after modeling. The gastrocnemius muscles of all experimental groups showed a certain degree of muscle atrophy. Fig.12 As shown, the wet weight ratios between the experimental side and the healthy side in the APML@PC and Autograft groups were 62.87±4.91% and 82.87±3.04%, respectively, which were significantly higher than those in the PC group (19.34±2.59)% and APL@PC (45.45±6.27)%.

[0090] like Figure 5 B. Fig.13 As shown, in order to further analyze the atrophy of the gastrocnemius muscle, the cross-section of the mid-lateral gastrocnemius muscle was stained with Masson's trichrome and the cross-sectional area of ​​the muscle fibers in each group was statistically analyzed. The results showed that compared with APL@PC and PC, the muscle fibers of APML@PC and Autograft were larger, more neatly arranged, more regular and orderly in shape, and the degree of atrophy was reduced.

[0091] Example 6

[0092] The sciatic nerve tissues of each group were fixed with 4% paraformaldehyde for 48 h, dehydrated with gradient sucrose (20%, 30%) until the tissue sank to the bottom, embedded in OCT, and frozen (cross-section and longitudinal section) into 10 μm tissue sections. The cross-sections were stained with toluidine blue (TB) and immunofluorescence staining (mouse anti-NF200 (1:100) and rabbit anti-MBP (1:100)), and the longitudinal sections were immunofluorescence staining (mouse anti-NF200 (1:100) and rabbit anti-S100β (1:100)). After incubation overnight, the sections were incubated with goat anti-rabbit Alexa Fluor®594 (1:200) and goat anti-mouse Alexa Fluor®488 (1:200), respectively. Finally, the cell nuclei were counterstained and sealed with anti-fluorescence attenuation mounting medium (containing DAPI), and observed under an upright fluorescence microscope or laser confocal microscope.

[0093] like Figure 6 , Fig.14 As shown, immunofluorescence staining of cross-sections showed the effects of nerve conduits in each group on axon regeneration and myelination. 12 weeks after surgery, nearly half of the synapses (NF200, green) in the APML@PC and Autograft groups were wrapped by myelin (MBP, red), indicating that these synapses were remyelinated. Compared with the APL@PC and PC groups, the APML@PC group significantly enhanced the axon regeneration and myelination ability of the sciatic nerve.

[0094] Example 7

[0095] The distal end (1-2 mm) of each regenerated nerve was collected. 3 ) in precooled electron microscopy fixative and fixed overnight in a 4°C refrigerator. Fixation with 1% osmium acid, dehydration with gradient alcohol, embedding, sectioning (80 nm), staining, and transmission electron microscopy (TEM) observation were performed. Ten photos were taken for each sample, and the average diameter of myelin sheath and the inner area of ​​axons / outer area of ​​myelinated fibers in each group of samples were quantified by Image J.

[0096] like Figure 7As shown, 12 weeks after surgery, the regenerated nerve fiber bundles in APML@PC contained a large number of myelinated axons and a high density of Nissl bodies, without excessive scars or adipose tissue, which was superior to the APL@PC and PC groups.

[0097] like Fig.15 , Fig.16 , Fig.17 As shown, TEM was used to further observe and quantify the axon diameter and the ratio of the inner axon area to the outer myelin area at the distal end of the regenerated nerve in each group. The APML@PC and Autograft groups contained more remyelinated axons and fewer vacuoles, and the axon diameter and the ratio of the inner axon area to the outer myelin area of ​​the regenerated nerve were significantly better than those of the other two groups. Therefore, compared with APL@PC and PC, APML@PC can effectively repair damaged nerves and promote axon remyelination.

[0098] Although the present invention is described herein with reference to a number of illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, drawings, and claims, a variety of variations and modifications may be made to the components and / or layout of the subject combination layout. In addition to the variations and modifications made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. An electrospun nanofiber double-layer nerve conduit, characterized in that: The double-layer nerve conduit is APML@PC, which is composed of PVA-CMCS and PCL-MoS2-LN electrospinning membranes to form a double-layer nerve conduit; The PVA-CMCS is polyvinyl alcohol-carboxymethyl chitosan, and the PCL-MoS2-LN is polycaprolactone-molybdenum disulfide cross-linked laminin; APML@PC is set as a catheter structure, the inner layer of the APML@PC catheter is PCL-MoS2-LN electrospinning membrane, and the outer layer of the APML@PC catheter is PVA-CMCS; Application of APML@PC as a nerve conduit for the treatment of peripheral nerve injury.

2. The electrospun nanofiber double-layer nerve conduit according to claim 1, characterized in that: The preparation method of the electrospun nanofiber double-layer nerve conduit comprises the following steps: Step 1) Preparation of inner layer: MoS2 nanosheet powder is added to HFIP solution and ultrasonically obtained into a dispersion, and then PCL is added into the dispersion and stirred to obtain a spinning solution; The spinning solution is prepared into an ordered fiber membrane PCL-MoS2 in an electrospinning machine, and the ordered fiber membrane PCL-MoS2 is added into a MES / EDC / NHS solution, incubated at room temperature, and then immersed in LN and shaken to obtain an ordered fiber membrane of cross-linked LN, namely, a PCL-MoS2-LN electrospinning membrane; Step 2) Preparation of outer layer: PVA and deionized water are mixed and heated and stirred to obtain a PVA solution, and then CMCS is added and stirred. After stirring, the solution is injected into a mold, and a stainless steel stick is inserted in the middle to seal the two ends of the mold. The solution is frozen and thawed, and demolded to obtain a nerve conduit PVA-CMCS. Step 3) Preparation of double-layer nerve conduit: The PCL-MoS2-LN electrospun membrane was cut into rectangles, rolled up with a stainless steel stick and inserted into the PVA-CMCS nerve conduit to prepare the double-layer nerve conduit APML@PC.

3. The electrospun nanofiber double-layer nerve conduit according to claim 2, characterized in that: In the step 1), the concentration of the LN solution is 0.2-2 mg / mL.

4. The electrospun nanofiber double-layer nerve conduit according to claim 2, characterized in that: In the step 2), the mold is a polyethylene tetrafluoroethylene tube mold, and the length of the polyethylene tetrafluoroethylene tube mold is 0.5-5 cm and the inner diameter is 1-3 mm.

5. The electrospun nanofiber double-layer nerve guide according to claim 2, characterized in that: In the step 2), a stainless steel stick with a diameter of 0.5-2.5 mm is inserted in the middle.

6. The electrospun nanofiber double-layer nerve conduit according to claim 2, characterized in that: In the step 3), the PCL-MoS2-LN electrospun membrane is cut into a rectangle with a length of 6-24 mm and a width of 2.5-10 mm.

7. The electrospun nanofiber double-layer nerve guide according to claim 2, characterized in that: In step 3), a stainless steel stick with a diameter of 0.5-2.5 mm is used to roll up and insert into the single-layer nerve conduit.

8. The electrospun nanofiber double-layer nerve guide according to claim 2, characterized in that: In the step 2), the freezing and thawing cycles are 4-8 times.

9. The electrospun nanofiber double-layer nerve conduit according to claim 1, characterized in that: The APML@PC was immersed in PBS and then shaken, taken out after 2-12 weeks, freeze-dried, and weighed to calculate the in vitro weight loss rate.

10. The electrospun nanofiber double-layer nerve guide according to claim 1, characterized in that: The APML@PC is cut and sterilized, and then added to a physiological saline warm water bath for incubation and set aside; Fresh blood of rats was anticoagulated with sodium citrate and centrifuged to separate red blood cells. After repeated washing with physiological saline, a red blood cell suspension was prepared. The cell suspension was aspirated and incubated in the reserved APML@PC. After incubation, physiological saline was added to stop hemolysis. The incubation was continued at a constant temperature. After the incubation, the blood was centrifuged and the supernatant was taken to detect the amount of hemoglobin released and the hemolysis rate was calculated.

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