Bionic multi-channel composite nerve conduit with viscidity and piezoelectric stimulation and preparation method of bionic multi-channel composite nerve conduit

Abstract

The invention discloses a bionic multi-channel composite nerve conduit with viscidity and piezoelectric stimulation and a preparation method of the bionic multi-channel composite nerve conduit. PAAM / SA / HA hydrogel developed by a one-pot method is used as an outer layer, and an Nrg-1 atSF / CNTs electrostatic spinning fiber conduit obtained by an electrostatic spinning technology is combined as an inner layer, so that the composite nerve conduit with an inner-outer structure is formed. Wherein the PAAM / SA / HA hydrogel internally contains a large number of hydrogen bonds, so that the PAAM / SA / HA hydrogel has super-strong viscosity, can replace surgical sutures, and also can inhibit scar formation; the Nrg-1 (at) SF / CNTs electrostatic spinning fiber catheter can be used as an orientation physical clue to enhance directional migration of nerve cells and induce directional growth of axons, and can also be used as a piezoelectric stimulation platform to realize controllable release of Nrg-1 to promote dynamic immune regulation of a nerve defect microenvironment and promote formation of myelin sheaths, and a new strategy is provided for nerve regeneration.

Description

Technical Field

[0001] The invention belongs to the technical field of medical supplies, and in particular relates to a bionic multi-channel composite nerve conduit with viscosity and piezoelectric stimulation and a preparation method thereof. Background Art

[0002] Peripheral nerve injury (PNI) is a common neurosurgical disease caused by acute trauma, which can lead to partial loss of sensory or motor function, or even disability. Autologous nerve transplantation is currently the recognized "gold standard" for repairing peripheral nerve defects in clinical practice, but the drawbacks of donor nerve deficiency, secondary trauma, mismatched nerve size, and neuroma formation limit the widespread application of autologous nerve transplantation. Cell replacement therapy has become a major clinical method for repairing peripheral nerve defects because of its low immune rejection reaction and high biological activity. However, the inevitable high shear stress and high oxidative stress in cell replacement therapy often cause reduced cell activity and significant functional differences between newborn nerves and surrounding normal tissues, resulting in low repair efficiency and poor regeneration effect. Tissue-engineered nerve grafts are an innovative product that combines cell culture technology and biomaterials science, designed to provide mechanical protection for nerve tissue in the lumen and provide growth space and necessary physical and biochemical cues for regenerated axons. The research and development of tissue-engineered nerve grafts with high repair efficiency is a hot topic of common concern in multiple disciplines such as materials science, tissue engineering, and medicine, and is of great significance to improving the quality of life of the people.

[0003] The present invention constructs a PAAM / SA / HA interpenetrating viscous network through a one-pot preparation technology, which can effectively inhibit scar hyperplasia, and combines electrospinning technology to construct a directional orientation structure, providing physical clues for improving the optimal directional extension of nerve axons, and it can be used as a piezoelectric stimulation platform, that is, the carboxyl end of CNTs is connected with Nrg-1 through electrostatic interaction, and converted into an electrical signal under the action of pressure, stimulating the controllable release of Nrg-1, thereby promoting peripheral nerve regeneration and immunomodulating the microenvironment, realizing precise dynamic regulation of the key links of nerve regeneration, and is expected to become a potential solution for repairing peripheral nerve defects in clinical practice. Summary of the invention

[0004] The present invention aims to provide a multi-channel composite nerve conduit with bionic viscosity and piezoelectric stimulation and a preparation method thereof. The composite nerve conduit has strong viscosity, can avoid suturing and inhibit scar hyperplasia, and can immunoregulate the nerve regeneration microenvironment through the controllable release of Nrg-1 in response to piezoelectric stimulation, and is ultimately used for high-efficiency repair of long-distance peripheral nerve defects, and is expected to achieve peripheral nerve regeneration.

[0005] To achieve the above object, the present invention adopts the following technical solution: A bionic multi-channel composite nerve conduit with viscosity and piezoelectric stimulation, which is composed of a PAAM / SA / HA hydrogel as an outer layer and a plurality of Nrg-1@SF / CNTs electrospun fiber conduits as an inner layer.

[0006] The preparation method of the composite nerve conduit comprises the following steps: (1) dissolving acrylamide solid (PAAM) in deionized water and stirring evenly at room temperature to obtain a PAAM solution; (2) dissolving sodium alginate solid (SA) in deionized water and stirring evenly at room temperature to obtain a SA solution; (3) dissolving hyaluronic acid solid (HA) in deionized water and stirring evenly at room temperature to obtain a HA solution; (4) Mix the PAAM solution and the SA solution to obtain the S1 solution; add the HA solution to the S1 solution to obtain the S2 solution; add N,N' -methylenebisacrylamide (MBAA) and tetramethylethylenediamine (TEMED) to obtain S3 solution; ammonium persulfate (APS) was added to the S3 solution to obtain PAAM / SA / HA hydrogel; (5) dissolving silk fibroin (SF) in formic acid to obtain S4 solution; adding carboxylated carbon nanotubes (CNTs) to the S4 solution to obtain S5 solution; subjecting the S5 solution to electrospinning to obtain an oriented electrospun membrane SF / CNTs; (6) rolling the electrospun membrane SF / CNTs obtained in step (5) into a tube shape, placing it in a neuregulin (Nrg-1) solution for ultrasonic treatment, and obtaining an electrospun fiber tube Nrg-1@SF / CNTs loaded with Nrg-1; (7) Wrapping the PAAM / SA / HA hydrogel obtained in step (4) on the outside of several electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6) to obtain the composite nerve conduit.

[0007] Furthermore, the mass concentration of the PAAM solution obtained in step (1) is 10% to 30%.

[0008] Furthermore, the mass concentration of the SA solution obtained in step (2) is 1% to 10%.

[0009] Furthermore, the mass concentration of the HA solution obtained in step (3) is 1% to 10%.

[0010] Furthermore, in the S1 solution obtained in step (4), the volume ratio of the SA solution to the PAAM solution is (0.1-0.5):1.

[0011] Furthermore, the volume ratio of HA solution to PAAM solution in the S2 solution obtained in step (4) is (0.01~0.1):1.

[0012] Furthermore, the mass ratio of MBAA, TEMED, APS and PAAM used in step (4) is (0.005~0.1):(0.01~0.1):(0.01~0.1):1.

[0013] Furthermore, the mass concentration of SF in the S4 solution obtained in step (5) is 5% to 30%.

[0014] Furthermore, the mass ratio of CNTs to SF in the S5 solution obtained in step (5) is (0.01-0.1):1.

[0015] Furthermore, in step (5), the temperature of the electrospinning treatment is 15-50°C, the humidity is 40-60%, the voltage is 5-25 kV, the receiving distance is 5-20 cm, and the drum speed is 500-3000 rpm.

[0016] Furthermore, the concentration of the Nrg-1 solution used in step (6) is 0.1-2 μg / mL.

[0017] Furthermore, the power of the ultrasonic treatment in step (6) is 400-900 W, and the time is 0.5-4 h.

[0018] The present invention provides a bionic multi-channel composite nerve conduit with viscosity and piezoelectric stimulation and a preparation method thereof. The bionic multi-channel composite nerve conduit is constructed by a one-pot method and an electrospinning fiber conduit is constructed in combination with electrospinning technology. Meanwhile, the interpenetrating double network structure of the hydrogel can be adjusted by regulating parameters such as acrylamide concentration, crosslinking degree and crosslinking time. The piezoelectric response stimulation performance of the electrospinning membrane can be regulated by changing factors such as the composition ratio of SF / CNTs, the concentration of CNTs and the mass fraction of formic acid. In addition, Nrg-1 loaded by electrostatic interaction at the carboxyl end of CNTs can convert the mechanical force generated by the contraction of the nerve itself and muscle compression into an electrical signal through piezoelectric stimulation, thereby realizing the controllable release of the neuromodulator protein Nrg-1, and further promoting immune regulation of the nerve defect microenvironment, promoting axon extension and myelin regeneration, and finally obtaining a series of multi-channel composite scaffolds that meet the staged needs of tissue repair.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The PAAM / SA / HA hydrogel prepared by the present invention has super strong viscosity due to the large amount of hydrogen bonds contained inside. It can not only replace surgical sutures to avoid inflammatory reactions caused by sutures, but also inhibit scar hyperplasia.

[0020] (2) The electrospinning catheter prepared by the present invention has good mechanical properties and anisotropy, can prevent axon dispersion, reduce nerve mismatch, and is beneficial to the recovery of nerve function. It can also provide physical clues for enhancing the directional extension of nerve regeneration, meet the requirements of long-distance nerve regeneration, and achieve long-term directional chemotaxis and guidance of nerve growth. It can also controllably release Nrg-1 through piezoelectric stimulation, promote peripheral nerve regeneration, and dynamically regulate the peripheral nerve regeneration microenvironment to achieve immune regulation.

[0021] (3) The present invention is based on hydrogen bond adhesion, piezoelectric stimulation controlled release of Nrg-1, and immune regulation of the regenerative microenvironment, which can comprehensively improve the efficiency of long-distance peripheral nerve defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Comparison diagram of the PAAM prepared in Comparative Example 1 (left) and the PAAM / SA / HA (right) hydrogel prepared in Example 3: wherein A is a scanning electron microscope image of the cross section of different hydrogels; B is a pore size distribution diagram of different hydrogels; C is an infrared spectrum diagram of different hydrogels; D is a tensile curve of different hydrogels; E is a compression curve of different hydrogels; F is a cycle curve of different hydrogels after 50 compressions, G is the tensile strength and fracture strain of different hydrogels; H is the compression modulus and compression strength of different hydrogels; I is the swelling curve of different hydrogels; and J is the degradation curve of different hydrogels.

[0023] Figure 2 Characterization diagram of the adhesion performance of the PAAM / SA / HA hydrogel prepared in Example 3: wherein A is the shear adhesion strength of different hydrogels on different substrates (pigskin, wood, nylon); B is the shear adhesion strength of the PAAM / SA / HA hydrogel on different substrates (pigskin, wood, nylon) and at different times (0 h, 5 h); C is the shear adhesion strength of the PAAM / SA / HA hydrogel after repeated adhesion to pigskin for 10 times; D is the adhesion picture of the PAAM / SA / HA hydrogel in different substrates (rubber, glass, pigskin, wood, plastic, cardboard, foam and iron); E is a diagram of the adhesion mechanism of the PAAM / SA / HA hydrogel.

[0024] Figure 3The comparison diagram of the electrospun fiber conduit Nrg-1@SF / CNTs prepared in Example 3 and the electrospun fiber conduit SF prepared in Comparative Example 2: A is the preparation flow chart of the electrospun fiber conduit Nrg-1@SF / CNTs; B and C are scanning electron microscope images of SF and SF / CNTs fiber membranes, respectively; D and E are the fiber diameter distributions of SF and SF / CNTs fiber membranes, respectively; F and G are the fiber orientation distributions of SF and SF / CNTs fiber membranes, respectively; H and I are the fiber orientation distributions of SF and SF / CNTs fiber membranes, respectively. Fiber tensile curve of NTs fiber membrane; J and K are the quantitative data of fiber tensile strength and strain of SF and SF / CNTs fiber membrane, respectively; L is the Fourier infrared spectrum of SF and SF / CNTs fiber membrane; M is the degradation diagram of SF and SF / CNTs fiber membrane; N is the XRD of SF and SF / CNTs fiber membrane; O is the piezoelectric open circuit voltage curve of SF / CNTs fiber membrane; P is the standard curve of free Nrg-1 release; Q is the Nrg-1 release curve of Nrg-1@SF / CNTs fiber membrane.

[0025] Figure 4 Characterization diagram of the PAAM / SA / HA / Nrg-1@SF / CNTs composite nerve conduit prepared in Example 3: wherein A is a scanning electron microscope image of the composite nerve conduit; B is an inverted microscope image of the interface of the composite nerve conduit; C is the piezoelectricity of the composite nerve conduit; D is an interface peeling curve graph of the composite nerve conduit; E is an interface shear adhesion curve graph of the composite nerve conduit; F is an interface adhesion curve graph of the composite nerve conduit; G is the quantitative data of interface peeling adhesion; H is the quantitative data of interface shear adhesion; and I is the quantitative data of double-layer interface adhesion.

[0026] Figure 5 Characterization diagram of the biocompatibility of the composite nerve conduits obtained in Example 3 and Comparative Examples 1 and 2: Live and dead cell immunofluorescence staining data of four groups of different scaffolds (blank group, PAAM / SA / HA / Nrg-1@SF / CNTs group, PAAM / Nrg-1@SF / CNTs group, PAAM / SA / HA / SF group) after co-culture with PC12 cells for 24 h and 48 h. DETAILED DESCRIPTION

[0027] A bionic multi-channel composite nerve conduit with viscous and piezoelectric stimulation, the preparation of which comprises the following steps: (1) Dissolve solid acrylamide (PAAM) in deionized water and stir at room temperature for 10 to 30 min to make it uniform, to obtain a PAAM solution with a mass concentration of 10% to 30%; (2) dissolving sodium alginate solid (SA) in deionized water and stirring at room temperature for 1 to 12 h to make it uniform, to obtain a SA solution with a mass concentration of 1% to 10%; (3) Dissolve hyaluronic acid solid (HA) in deionized water and stir at room temperature for 12 to 48 hours to make it uniform, to obtain a HA solution with a mass concentration of 1% to 10%; (4) SA solution and PAAM solution were mixed in a volume ratio of (0.1-0.5):1 to obtain S1 solution; a certain amount of HA solution was added to S1 solution in a volume ratio of HA solution to PAAM solution of (0.01-0.1):1 to obtain S2 solution; N,N' -methylenebisacrylamide (MBAA) and tetramethylethylenediamine (TEMED) to obtain S3 solution; ammonium persulfate (APS) was added to the S3 solution to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM was (0.005-0.1):(0.01-0.1):(0.01-0.1):1; (5) Dissolving silk fibroin (SF) in formic acid to obtain a S4 solution with a mass concentration of 5% to 30%; adding carboxylated carbon nanotubes (CNTs) to the S4 solution at a mass ratio of (0.01 to 0.1):1 to obtain a S5 solution; subjecting the S5 solution to electrospinning to obtain an oriented electrospinning membrane SF / CNTs; the electrospinning temperature is 15 to 50 °C, the humidity is 40 to 60%, the voltage is 5 to 25 kV, the receiving distance is 5 to 20 cm, and the drum speed is 500 to 3000 rpm; (6) Using a needle, the electrospun SF / CNTs membrane obtained in step (5) was rolled into a tube shape, placed in a 0.1-2 μg / mL neuregulin (Nrg-1) solution, and treated at an ultrasonic power of 400-900 W for 0.5-4 h to obtain an electrospun fiber tube Nrg-1@SF / CNTs loaded with Nrg-1; (7) The PAAM / SA / HA hydrogel obtained in step (4) is spread on a glass plate, and several electrospun fiber conduits obtained in step (6) are placed on the surface of the hydrogel so that the hydrogel is completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a bionic multi-channel composite nerve conduit.

[0028] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0029] Example 1 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 10%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0030] Example 2 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 10%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 5% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0031] Example 3 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0032] Example 4 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 5% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / VThe solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0033] Example 5 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 15% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0034] Example 6 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 20% W / V solution; CNTs were added to the solution at a mass ratio of CNTs to SF of 0.1:1, and the solution was stirred evenly for electrospinning at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospinning membrane SF / CNTs with a thickness of about 1 mm; (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0035] Example 7 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 1000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0036] Example 8 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 3% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 500 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (6) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the obtained electrospun membrane SF / CNTs into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (7) The PAAM / SA / HA hydrogel obtained in step (4) was spread on a glass plate, and several electrospun fiber conduits obtained in step (6) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6), thereby obtaining a PAAM / SA / HA / Nrg-1@SF / CNTs biomimetic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0037] Comparative Example 1 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) adding MBAA, TEMED and APS to the PAAM solution and mixing them evenly to obtain a PAAM hydrogel; wherein the mass ratio of the added MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (3) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The solution was prepared by adding CNTs to SF at a mass ratio of 0.02:1. The solution was stirred evenly and then electrospun at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane SF / CNTs with a thickness of about 1 mm. (4) Use a needle (syringe size is 1 mL, needle inner diameter is 1 mm) to roll the electrospun SF / CNTs membrane into a tube shape, put it into a 1 μg / mL Nrg-1 solution, and treat it under 600 W ultrasonic power for 1 h to obtain Nrg-1-loaded electrospun fiber tube Nrg-1@SF / CNTs; (5) The PAAM hydrogel obtained in step (2) was spread on a glass plate, and several electrospun fiber conduits obtained in step (4) were placed on the surface of the hydrogel so that the hydrogel was completely wrapped around the outside of the electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (5), thereby obtaining a PAAM / Nrg-1@SF / CNTs bionic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0038] Comparative Example 2 (1) Dissolve PAAM powder in deionized water and stir at room temperature for 30 min to obtain a PAAM solution with a mass concentration of 15%; (2) Dissolve SA powder in deionized water and stir at room temperature for 12 h to obtain a 5% SA solution; (3) Dissolve HA powder in deionized water and stir at room temperature for 48 h to obtain a HA solution with a mass concentration of 2%; (4) Stirring PAAM solution, SA solution and HA solution in a volume ratio of 1:0.15:0.01 at room temperature, and then adding MBAA, TEMED and APS, and mixing evenly to obtain PAAM / SA / HA hydrogel; wherein the mass ratio of MBAA, TEMED, APS to PAAM is 0.005:0.01:0.015:1; (5) Dissolve SF in formic acid and stir evenly to obtain a concentration of 10% W / V The obtained solution was subjected to electrospinning treatment at a voltage of 15 kV, a temperature of 30 °C, a humidity of 50%, a receiving distance of 15 cm, and a drum speed of 2000 rpm to obtain an oriented electrospun membrane with a thickness of about 1 mm; (6) Use a needle (syringe specification is 1 mL, needle inner diameter is 1 mm) to roll the electrospun membrane into a tube shape to obtain an electrospun fiber tube SF; (7) The PAAM / SA / HA hydrogel obtained in step (4) is spread on a glass plate, and several electrospun fiber conduits obtained in step (6) are placed on the surface of the hydrogel so that the hydrogel completely wraps the outside of the electrospun fiber conduits SF obtained in step (6) to obtain a PAAM / SA / HA / SF bionic viscous multi-channel composite nerve conduit with an inner diameter of 3 mm and an outer diameter of 5 mm.

[0039] Table 1 Effect of different PAAM / SA ratios on the shear adhesion strength of hydrogels

[0040] It can be seen from Table 1 that the shear adhesion strength of hydrogels with different PAAM / SA ratios is significantly different. After adjusting the experimental conditions, the appropriate PAAM mass concentration is finally determined to be 15% and the SA mass concentration is 3%.

[0041] Table 2 Effect of different rotation speeds on the orientation of electrospun fiber membranes

[0042] It can be seen from Table 2 that different rotation speeds have obvious differences on the orientation of electrospun fiber membranes. After adjusting the experimental conditions, the electrospinning speed was finally determined to be 2000 rpm, and the obtained electrospun fiber membrane had good orientation.

[0043] Table 3 Effect of different SF concentrations on the diameter of electrospun fiber membranes

[0044] It can be seen from Table 3 that the diameters of electrospun fiber membranes prepared with different SF concentrations are significantly different. After adjusting the experimental conditions, the SF concentration was finally determined to be 10%, and the diameter of the electrospun fiber membrane obtained was more in line with the requirements of the neural scaffold.

[0045] Figure 1 This is a comparison diagram of the PAAM / SA / HA prepared in Example 3 and the PAAM hydrogel prepared in Comparative Example 1. As can be seen from the figure, the PAAM / SA / HA hydrogel was successfully synthesized and has a microtubule array bionic microstructure, realizing the three-dimensional multi-channel microelement pore structure bionics. From the tensile, compression and cyclic compression diagrams, it can be seen that the PAAM / SA / HA hydrogel has good mechanical properties and cyclic stability. From the swelling and degradation diagrams, it can be seen that the PAAM / SA / HA hydrogel has certain swelling and degradability, which can meet the requirements of certain mechanical support and degradability for nerve regeneration.

[0046] Figure 2 This is a characterization diagram of the adhesion performance of the PAAM / SA / HA hydrogel prepared in Example 3. As can be seen from the figure, the addition of sodium alginate and hyaluronic acid can significantly enhance the adhesion performance of the hydrogel. The main reason is that sodium alginate and hyaluronic acid have a large number of carboxyl groups and hydroxyl groups, which can form hydrogen bonds with the amino groups in acrylamide to enhance the adhesion of the hydrogel. The enhanced adhesion of the PAAM / SA / HA hydrogel allows the outer layer of the hydrogel to better adhere and connect the nerve ends, avoiding surgical sutures and playing a role in inhibiting scars.

[0047] Figure 3 This is a comparison diagram of the electrospun fiber conduit Nrg-1@SF / CNTs prepared in Example 3 and the electrospun fiber membrane conduit SF prepared in Comparative Example 2. Figure 3 It can be seen that the prepared Nrg-1@SF / CNTs has good orientation and good mechanical properties. From XRD and infrared, it can be seen that SF has a β phase, which is piezoelectric, and the addition of CNTs does not affect the piezoelectricity of SF. Nrg-1 can be released for immune regulation. The voltage-current diagram further verifies that Nrg-1@SF / CNTs has piezoelectricity, and the release of Nrg-1 verifies its release responsiveness.

[0048] Figure 4 This is a characterization diagram of the PAAM / SA / HA / Nrg-1@SF / CNTs composite nerve conduit prepared in Example 3. As can be seen from the figure, the obtained composite nerve conduit has a multi-channel structure similar to that of natural nerves, and its hydrogel and electrospun fiber conduit interface are tightly adhered and still have good piezoelectricity.

[0049] The composite nerve conduits obtained in Example 3 and Comparative Examples 1 and 2 were co-cultured with PC12 cells for 24 h and 48 h, respectively. The live and dead cells were fluorescently stained using a live / dead cell double staining kit, and the number of live and dead cells was monitored under a fluorescence microscope to examine their biocompatibility. The results are shown in Table 1. Figure 5 As can be seen from the figure, the number of dead cells in different composite nerve conduits is almost negligible, indicating that they have good biocompatibility.

[0050] Table 4 Statistics of biological activity and neurite growth length of different composite nerve conduits

[0051] The cell viability was quantified, and the results are shown in Table 4. As can be seen from Table 4, the cell viability on different hydrogels can reach more than 90%. At the same time, the growth and migration of nerve axons were quantified by the extension length of the dorsal root ganglion on the hydrogel. It can be seen that comparative examples 1 and 2 lacked directional induction on the neurites, and the average length of the neurites after 48 hours was only 400~600µm, while Example 3 with directional multi-channels provided physical clues for the optimal directional extension of the nerve axons. After 48 hours, the average length of the nerve axons reached 1000~1100 µm, proving the directional chemotaxis and guidance effect of the hydrogel on the growth of axons.

[0052] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

[0053] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bionic multi-channel composite nerve conduit with viscous and piezoelectric stimulation, characterized in that: The composite nerve conduit is composed of a PAAM / SA / HA hydrogel as an outer layer, which wraps a plurality of Nrg-1@SF / CNTs electrospun fiber conduits as an inner layer.

2. A method for preparing a bionic multi-channel composite nerve conduit with viscous and piezoelectric stimulation as claimed in claim 1, characterized in that: The following steps are involved: (1) Dissolve PAAM in deionized water and stir evenly at room temperature to obtain a PAAM solution; (2) Dissolve SA in deionized water and stir evenly at room temperature to obtain a SA solution; (3) Dissolve HA in deionized water and stir evenly at room temperature to obtain a HA solution; (4) Mixing the PAAM solution with the SA solution to obtain the S1 solution; adding the HA solution to the S1 solution to obtain the S2 solution; adding MBAA and TEMED to the S2 solution to obtain the S3 solution; adding APS to the S3 solution to obtain the PAAM / SA / HA hydrogel; (5) dissolving silk fibroin in formic acid to obtain S4 solution; Add CNTs to the S4 solution to obtain the S5 solution; The S5 solution was subjected to electrospinning to obtain oriented electrospun membrane SF / CNTs; (6) rolling the electrospun membrane SF / CNTs obtained in step (5) into a tube shape, placing it in a Nrg-1 solution for ultrasonic treatment, and obtaining an electrospun fiber tube Nrg-1@SF / CNTs loaded with Nrg-1; (7) Wrapping the PAAM / SA / HA hydrogel obtained in step (4) on the outside of several electrospun fiber conduits Nrg-1@SF / CNTs obtained in step (6) to obtain the composite nerve conduit.

3. The preparation method according to claim 2, characterized in that: The mass concentration of the PAAM solution obtained in step (1) is 10% to 30%.

4. The preparation method according to claim 2, characterized in that: The mass concentration of the SA solution obtained in step (2) is 1% to 10%.

5. The preparation method according to claim 2, characterized in that: The mass concentration of the HA solution obtained in step (3) is 1% to 10%.

6. The preparation method according to claim 2, characterized in that: In the S1 solution obtained in step (4), the volume ratio of SA solution to PAAM solution is (0.1-0.5):1; the volume ratio of HA solution to PAAM solution in the S2 solution obtained is (0.01-0.1):1; the mass ratio of MBAA, TEMED, APS and PAAM used is (0.005-0.1):(0.01-0.1):(0.01-0.1):

1.

7. The preparation method according to claim 2, characterized in that The mass concentration of SF in the S4 solution obtained in step (5) is 5%~30%; the mass ratio of CNTs to SF in the S5 solution obtained is (0.01~0.1):1; the temperature of the electrospinning treatment is 15~50°C, the humidity is 40~60%, the voltage is 5~25 kV, the receiving distance is 5~20 cm, and the drum speed is 500~3000 rpm.

8. The preparation method according to claim 2, characterized in that The concentration of the Nrg-1 solution used in step (6) is 0.1-2 μg / mL; the power of the ultrasonic treatment is 400-900 W, and the time is 0.5-4 h.

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