Sensory / motor neuron differentiated neural restoration catheter based on controllable release of metal ions triggered by ultrasound

Abstract

The invention discloses a sensory / motor neuron differentiated neural restoration catheter based on controllable release of metal ions triggered by ultrasound. The SF / metal ion neural restoration conduit with an ultrasonic piezoelectric and directional topological structure is developed by combining a one-pot method with an electrostatic spinning process. Wherein the oriented structure of the SF spinning membrane can promote the directional migration of cells and the directional extension of axons while being physically guided; and the material can also be used as a stimulation response platform to generate piezoelectricity under the ultrasonic action, so that the controllable release of metal ions is realized. The released metal ions (such as Zn2, Cu2, K, Mg2 and the like) can accurately program cell behaviors by activating intracellular signal transduction pathways in sensory / motor neurons in the neural restoration process. Expression of neurotrophic factors and receptors of the neurotrophic factors can be differentially up-regulated or inhibited, and other possibilities are provided for differentiated regeneration of sensory / motor neurons.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials, and particularly relates to a sensory / motor neuron differential repair conduit based on ultrasound-triggered controlled release of metal ions and a preparation method thereof. BACKGROUND

[0002] Peripheral nerve injury (PNI) is one of the most common forms of trauma, which seriously affects the quality of life of patients and causes a large number of disabilities. Peripheral nerves show self-repair potential after mild and moderate trauma. However, when the length of peripheral nerve defect is greater than 5 mm, it is very difficult to rely on the nerve itself to achieve defect healing and functional recovery. It is worth noting that the nervous system achieves perception and response to internal and external environment through two types of functionally distinct peripheral projection neurons-sensory (afferent) neurons and motor (efferent) neurons. Sensory neurons convert mechanical, chemical, temperature and other stimuli into electrical signals to the center; motor neurons transmit central instructions to muscles or glands to produce contraction or secretion. Both of them show significant "differential" characteristics in developmental origin, molecular markers, synaptic plasticity and disease susceptibility, so accurate regulation of them has become a core requirement for nerve repair.

[0003] In view of this, artificial nerve guide conduit (NGC) has become a promising treatment strategy to promote peripheral nerve regeneration and functional recovery. NGC can bridge long distance nerve defects, provide mechanical support and favorable tissue regeneration microenvironment for axon regeneration and nerve repair, and ultimately help restore sensory and motor function. Electrical signals, as a medium for communication between cells, play an important role in nerve growth. Patent CN 117482282A discloses a double-layer intelligent nerve conduit of polyvinylidene fluoride-trifluoroethylene [P(VDF-TrFE)] with barium titanate (BaTiO3) and other inorganic piezoelectric nanoparticles planted by electrospinning process. However, the therapeutic factors released by the nerve conduit are limited to neurotrophic factors, ignoring the metal ions which play a key role in neural regulation, and the release process depends on the thermal expansion and contraction of the conduit itself, with limited regulation accuracy. Another patent CN107779672B discloses a nerve repair conduit doped with degradable zinc alloy, which promotes nerve repair through slow release of zinc ions (Zn 2 ⁺). However, the release of metal ions described in this patent completely depends on the degradation process of the material itself, and cannot be accurately regulated at a specific time window or spatial site, nor can it match the complex dynamic needs of nerve regeneration. Therefore, the present application aims to provide an intelligent nerve conduit that can accurately control the timing and dosage of metal ion release through external ultrasound stimulation, and can differentially regulate sensory and motor nerves according to their different physiological characteristics, in order to solve the problems of uncontrollable release of therapeutic factors and lack of specificity in repair strategies in the prior art.

[0004] In the nervous tissue, metal ions act as cofactors in proteome, playing a vital role in biological organisms by participating in various biological processes such as catalytic activity, signal transduction, cell metabolism and homeostasis. For example, after nerve injury, zinc ions can be released from specific vesicles of neurons or glial cells, or flow into the cytoplasm by up-regulation of zinc transporters (such as Zip family). The increase of intracellular zinc concentration can activate various kinases such as Raf and MEK, thereby enhancing the activity of the MAPK / ERK signaling pathway, which is a classic pathway for cell proliferation, differentiation and survival, and then promoting the expression of neurotrophic factors (such as BDNF, GDNF) and related regeneration genes, creating a favorable intracellular environment for axon regeneration. Magnesium ions (Mg 2+ ) provide the most basic energy supply for the whole nerve repair process by ensuring the effective use of ATP, thereby synthesizing new proteins and lipids and rebuilding ion gradients. In addition, copper ions (Cu 2+ ) are key cofactors for various oxidases, participate in antioxidant and connective tissue formation, effectively scavenge free radicals by ensuring the activity of SOD, and protect neurons and supporting cells from oxidative damage. These metal ions promote the coordinated development and maintenance of normal nervous system function by regulating signal transduction, enzyme activity and metabolic processes.

[0005] The application provides a sensory / motor neuron differential nerve repair conduit based on ultrasound-triggered controlled release of metal ions. The conduit is integrally formed by an innovative one-pot method combined with an electrospinning process, and a highly directional micro-nano topological structure is successfully constructed in the fiber membrane by precisely regulating the rotation speed parameter in the spinning process. The oriented structure not only can serve as a physical clue for guiding the directional extension of nerve axons, effectively promoting the adhesion, arrangement of Schwann cells and directional migration of neurons. More importantly, the conduit itself can respond to external ultrasound stimulation. When external ultrasound field is applied, SF undergoes conformational transition due to its inherent piezoelectric properties and ultrasound-induced mechanical vibration, and the proportion of beta-sheet conformation is significantly enhanced, thereby generating a controllable piezoelectric signal in the material interior. The piezoelectric effect can directly simulate the in vivo bioelectric environment on one hand, and produce an electric stimulus on nerve cells; on the other hand, it cooperatively triggers the on-demand and controllable release of metal ions from the fiber matrix. The specific metal ions (such as Zn 2 ⁺, Mg 2 ⁺, etc.) released in the nerve regeneration microenvironment play multiple key roles: they not only can directly participate in synaptic transmission and regulate neural plasticity, but also can activate different signal pathways (such as Ca 2+ channels, TRPA1 receptors, etc.) and downstream cytokine expression, achieving precise differential regulation of the regeneration of the two types of nerves. Finally, the nerve conduit synergistically promotes the effective regeneration of axons and the regular formation of myelin sheaths in space-time through the triple synergy of physical topology, piezoelectric electric stimulation, and metal ion chemical signals. In summary, the SF / metal ion chloride nerve conduit prepared by the present application has excellent biocompatibility, degradability, biomimetic structure, and active regulation ability, and exhibits significant peripheral nerve repair efficiency, providing a new and highly transformative strategy for solving the problem of clinical peripheral nerve defect repair. SUMMARY

[0006] The present application aims to provide a sensory / motor neuron differential nerve repair conduit based on ultrasound-triggered controlled release of metal ions, which provides a new idea for realizing peripheral nerve regeneration and broadens the application of piezoelectric materials in the field of nerve regeneration. The piezoelectric electrospun film in the nerve conduit can transmit electrical signals throughout the nerve conduit, and its oriented structure has a positive effect on the adhesion and directional migration of Schwann cells. The beta-sheet conformation of SF increases after soaking in organic solvents, and the piezoelectric performance is significantly improved. The nerve conduit has excellent mechanical properties and can support the entire process of nerve regeneration; and under the action of ultrasound, the metal ions are controllably released during nerve regeneration, which can regulate sensory neuron / myelin regeneration, respectively, to promote nerve regeneration. In summary, the present application creatively prepares a piezoelectric nerve conduit based on SF, which realizes the controlled release of metal ions through ultrasound triggering, thereby providing a new active regulation strategy for the differential and precise repair of sensory / motor nerves.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The sensory / motor neuron differential nerve repair conduit based on ultrasound-triggered controlled release of metal ions is composed of a piezoelectric SF electrospun film doped with metal ions.

[0008] The preparation method of the nerve conduit comprises the following steps: (1) Put the silk into the deionized water boiled in advance, add anhydrous sodium carbonate for degumming treatment to obtain silk fibroin (SF), and after drying, add lithium bromide or calcium chloride / anhydrous ethanol / water ternary solution for dissolution, dialysis, and centrifugation, filtration to obtain S1 solution.

[0009] (2) Freeze the S1 solution, and vacuum dry to obtain S2 solid.

[0010] (3) Dissolve the S2 solid in an organic solvent, then add a chloride containing metal ions, and after stirring, the metal ions can be combined with SF based on metal coordination bonds to obtain S3 solution.

[0011] (4) The S3 solution is treated by an electrospinning process, and relevant parameters are controlled to obtain an oriented electrospinning film S4.

[0012] (5) The electrospinning film S4 obtained in step (4) is soaked in an organic solvent, taken out, dried, and rolled into a conduit shape to obtain the nerve repair conduit. The soaking method of the organic solvent enables the nerve repair conduit to realize controllable release of metal ions in response to different degrees of ultrasonic stimulation.

[0013] Preferably, in step (1), the molar ratio of lithium bromide or calcium chloride, anhydrous ethanol, and water in the lithium bromide or calcium chloride / anhydrous ethanol / water ternary solution is 1:2:8.

[0014] Preferably, in step (2), the freezing treatment method is liquid nitrogen treatment or storage in a refrigerator, and the vacuum drying time is 1-4 days.

[0015] Preferably, in step (3), the organic solvent is one or two of chloroform, dichloromethane, hexafluoroisopropanol, and methanol; and the mass percentage concentration of SF in the obtained polymer solution (S3 solution) is 5.0%-20.0%.

[0016] Preferably, in step (3), the metal ion chloride is one of zinc chloride, potassium chloride, magnesium chloride, calcium chloride, and copper chloride, and the concentration of metal ions in the S3 solution is 0.1-20 mM.

[0017] Preferably, in step (5), the temperature of electrospinning is 15-50 ℃, the voltage is 5-25 kV, the receiving distance is 5.0-20.0 cm, and the roller rotation speed is 500-3000 rpm.

[0018] Preferably, in step (5), the organic solvent is one or two of methanol, ethanol, and propanol.

[0019] Preferably, in step (5), the drying temperature is 20-60 ℃, the drying time is 1.0-24.0 h, and the soaking time of the organic solvent is 1.0-12.0 h.

[0020] Further, in step (5), the different degrees of ultrasonic stimulation include: the ultrasonic time is 0.5-1.0 h, the ultrasonic stimulation power is 0.5-3.0 W / cm 2 , and the stimulation distance is 0.5-4.0 cm.

[0021] The SF / metal ion nerve conduit prepared in the application can realize directional guiding effect, can also promote axon regeneration and myelination through differential regulation of sensory neuron and motor neuron regeneration, finally effectively improves the repair efficiency of long peripheral nerve defect, and further helps the target organ function reconstruction.

[0022] Compared with the prior art, the application has the following beneficial effects: (1) The electrospun conduit prepared in the application has good mechanical properties and anisotropy, can prevent axon dispersion and reduce nerve mismatch, and the application does not simply superimpose single function, but creates multiple synergistic effects. First, the piezoelectric effect generates endogenous electric stimulation under ultrasound, simulates the physiological electrical activity of nerves, and directly promotes neuronal excitation and axon directional growth. Second, the controllably released metal ions act as key chemical signals to selectively regulate the gene expression and myelin synthesis of sensory or motor neurons. Finally, the oriented topological structure of the electrospun film physically guides the directional migration of Schwann cells and axons. The three (electricity, chemistry, and physical topology) synergistically act under the core means of ultrasound to construct a highly biomimetic and efficient regeneration-promoting composite microenvironment.

[0023] (2) By taking ultrasound stimulation as an external trigger switch, the timing, dose and space of metal ion release are accurately controlled. This overcomes the passivity and blindness of ion release depending on the spontaneous degradation of materials in the prior art, and can actively intervene by adjusting the ultrasound parameters (such as intensity, frequency, and duration) according to the needs of different stages of nerve regeneration and specific nerve types, to realize personalized and precise treatment, and significantly improve the repair efficiency and reliability.

[0024] (3) The application differentially regulates the axon regeneration and myelination of sensory / motor neurons, and simultaneously promotes the regeneration of motor nerves by Zn 2 ⁺, or the repair of sensory nerves by Mg 2 ⁺, etc., uses ultrasound for targeted regulation, and accurately repairs different components in mixed nerve injury. This solves the industry pain point that the repair effect of existing general nerve conduits is single and cannot consider different nerve function reconstruction, and provides a revolutionary tool for realizing the complete recovery of peripheral nerve function (including sensation and movement). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The morphology and structure characterization diagram of the electrospun film SF prepared in Example 9: A. Scanning electron microscope morphology diagram; B. Fiber diameter of the fiber film loaded with different ions; C. Successful loading of different metal ions and content; D. Tensile curve of the SF / Mg 2+ fiber film in different directions; E. SF / Mg 2+A. Tensile stress and Young's modulus of the fiber membrane in different directions; B. Hydrophilicity of the fiber membrane; C. SF / Mg 2+ Infrared spectrum of the fiber membrane.

[0026] Figure 2 Electrospun membrane SF / Mg prepared for Example 9 2+ Performance characterization graph: A. XRD of pure SF and SF after immersion in organic solvent methanol for 5h; B, C. SF / Mg 2+ Butterfly curve of the electrospun membrane; D. SF / Mg 2+ Ultrasonic response piezoelectricity graph of the electrospun membrane; E-H. SF / Mg 2+ Electrospun membrane d 33 And phase diagram; I, J. SF / Mg 2+ Voltage graph of the electrospun membrane at different distances / frequencies; K, L. SF / Mg 2+ Current graph of the electrospun membrane at different distances / frequencies.

[0027] Figure 3 Short-term and long-term release behavior performance characterization graph of metal ions in the electrospun membrane SF prepared for Example 9: A-C. Standard curves of different metal ions; D-F. Ultrasonic response short-term release behavior of different metal ions (ultrasonic frequency 2.5 W / cm 2 , distance 0.25 cm); G-I. Ultrasonic response long-term release behavior of different metal ions (ultrasonic frequency 2.5 W / cm 2 , distance 0.25 cm).

[0028] Figure 4 Influence characterization graph of metal ions in the electrospun membrane SF prepared for Example 11 on PC12 cell proliferation behavior at the cell level: A and C. Dead and live images of cell proliferation at three concentrations of magnesium ions, zinc ions for 1, 3, 7 days; B and D. Dead and live images of cell proliferation at three concentrations of calcium ions, copper ions for 1, 3, 7 days.

[0029] Figure 5 Influence characterization and quantification graph of metal ions in the electrospun membrane SF prepared for Example 11 on PC12 cell proliferation behavior at the cell level: A. Dead and live images of cell proliferation at three concentrations of potassium ions for 1, 3, 7 days; B-F. CCK8 column chart of co-culturing five kinds of metal ions (Zn 2+ , Cu 2+ , K + , Mg 2+ , Ca 2+ ) with PC12 cells for 1, 3, 7 days.

[0030] Figure 6The mechanism of electrospun film SF prepared in Example 9 promotes cell proliferation and promotes the regeneration of sensory / motor neurons at the cell level, wherein NGF is a specific growth factor in sensory / motor neurons, and different colored round balls represent different types of metal ions. DETAILED DESCRIPTION

[0031] The application discloses a preparation method of a sensory / motor neuron differential nerve repair conduit based on ultrasound-triggered controlled release of metal ions, which comprises the following steps: (1) Put silk into deionized water boiled in advance, add anhydrous sodium carbonate for degumming treatment, after drying, add lithium bromide or calcium chloride / anhydrous ethanol / water ternary solution for dissolution, dialysis, and centrifugation and filtration to obtain an SF solution; (2) Freeze the SF solution, and vacuum dry to obtain SF solids; (3) Dissolve 2 g of the SF solids in dichloromethane to obtain a high polymer solution with a mass percentage concentration of 10%; (4) Add metal ion chloride powder to the high polymer solution obtained in step (3) to obtain a spinning solution; (5) Under the condition of 15-50 ℃ and a voltage of 5-25 kV, the spinning solution obtained in step (4) is treated by an electrospinning process (the receiving distance is 5-20 cm, and the roller rotating speed is 800-3000 rpm) to obtain an electrospun film SF with piezoelectricity; (6) After the electrospun film SF obtained in step (5) is soaked in an organic solvent, it is dried at 20-60 ℃ for 5-30 min, and then is rolled into a tube.

[0032] In steps (3) and (6), the organic solvent is one or two of chloroform, dichloromethane, hexafluoroisopropanol, methanol and ethanol.

[0033] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described in combination with specific embodiments, but the application is not limited thereto.

[0034] Example 1 (1) Put 100 g of silk into 3 L of deionized water boiled in advance, add 20 g of anhydrous sodium carbonate for degumming treatment, after drying, add 240 mL of a calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water is 1:2:8) for dissolution, dialysis, and centrifugation and filtration to obtain an SF solution; (2) Freeze the SF solution, and vacuum dry to obtain SF solids; (3) Dissolve 2 g of the SF solids in dichloromethane to obtain a high polymer solution with a mass percentage concentration of 10%; (4) 20 mM of calcium chloride, potassium chloride, magnesium chloride or copper chloride powder was added into the polymer solution obtained in step (3); (5) The four polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30 °C and a voltage of 20 kV (a receiving distance of 10 cm and a drum rotating speed of 1000 rpm) to obtain four electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40 °C for 10 min, then soaked in methanol for 3 h, dried and rolled into tubes to obtain four nerve conduits.

[0035] Example 2 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, and after drying, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added for dissolution, dialysis, and centrifugation, filtration to obtain SF solution; (2) The SF solution was treated by freezing, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) 20 mM of calcium chloride, potassium chloride, magnesium chloride or copper chloride powder was added into the polymer solution obtained in step (3); (5) The four polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30 °C and a voltage of 20 kV (a receiving distance of 10 cm and a drum rotating speed of 1000 rpm) to obtain four electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40 °C for 10 min, then soaked in methanol for 3 h, dried and rolled into tubes to obtain four nerve conduits.

[0036] Example 3 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, and after drying, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added for dissolution, dialysis, and centrifugation, filtration to obtain SF solution; (2) The SF solution was treated by freezing, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders, calcium chloride, potassium chloride, magnesium chloride or copper chloride, were added to the polymer solution obtained in step (3), and the final molar concentration of the four kinds of metal ions was 20 mM; (5) The four kinds of polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30°C and a voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 1000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40°C for 10 min, then soaked in methanol for 8 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0037] Example 4 (1) 100 g of silk was placed in 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, and after drying, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added for dissolution, dialysis, and centrifugation, filtration to obtain an SF solution; (2) The SF solution was subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders, calcium chloride, potassium chloride, magnesium chloride or copper chloride, were added to the polymer solution obtained in step (3), and the final molar concentration of the four kinds of metal ions was 20 mM; 2+ , K + , Mg 2+ molar final concentration was 10 mM, and Cu 2+ molar final concentration was 100 μmM; (5) The four kinds of polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30°C and a voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 1000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40°C for 10 min, then soaked in methanol for 8 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0038] Example 5 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added after drying for dissolution, dialysis, and SF solution was obtained by centrifugation and filtration; (2) SF solid was obtained after vacuum drying after freezing treatment of the SF solution; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders of calcium chloride, potassium chloride, magnesium chloride or copper chloride were added to the high molecular solution obtained in step (3), wherein the molar final concentration of Ca 2+ , K + , Mg 2+ was 20 mM, and the molar final concentration of Cu 2+ was 100 μmM; (5) The four kinds of high molecular SF spinning solutions obtained in step (4) were treated by electrospinning process under the condition of 30°C and voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 1000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were dried at 40°C for 10 min, then soaked in methanol for 5 h to curl into tubes to obtain four kinds of nerve conduits.

[0039] Example 6 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added after drying for dissolution, dialysis, and SF solution was obtained by centrifugation and filtration; (2) SF solid was obtained after vacuum drying after freezing treatment of the SF solution; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders of calcium chloride, potassium chloride, magnesium chloride or copper chloride were added to the high molecular solution obtained in step (3), wherein the molar final concentration of Ca 2+ , K + , Mg 2+ was 20 mM, and the molar final concentration of Cu 2+ was 100 μmM; (5) The four kinds of high molecular SF spinning solutions obtained in step (4) were treated by electrospinning process under the condition of 30°C and voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 1000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun film obtained in step (5) is placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into a tube to obtain four nerve conduits.

[0040] Example 7 (1) 100 g of silk is placed in 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate is added for degumming treatment, after drying, 240 mL of lithium bromide or calcium chloride / absolute ethanol / water ternary solution (the molar ratio of calcium chloride, absolute ethanol and water is 1:2:8) is added for dissolution, dialysis, and centrifugation, filtration to obtain an SF solution; (2) The SF solution is subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid is dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders of calcium chloride or potassium chloride or magnesium chloride or copper chloride are added to the polymer solution obtained in step (3), and the molar final concentration of the four different metal ions is 5 mM; (5) The four kinds of polymer SF spinning solutions obtained in step (4) are subjected to electrospinning process under the condition of 30 °C and voltage of 20 kV (receiving distance is 10 cm, and the roller rotation speed is 1000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun film obtained in step (5) is placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into a tube to obtain four nerve conduits.

[0041] Example 8 (1) 100 g of silk is placed in 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate is added for degumming treatment, after drying, 240 mL of lithium bromide or calcium chloride / absolute ethanol / water ternary solution (the molar ratio of calcium chloride, absolute ethanol and water is 1:2:8) is added for dissolution, dialysis, and centrifugation, filtration to obtain an SF solution; (2) The SF solution is subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid is dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders of calcium chloride or potassium chloride or magnesium chloride or copper chloride are added to the polymer solution obtained in step (3), and the molar final concentration of the four different metal ions is 5 mM; 2+ , K + , Mg 2+ molar final concentration is 20 mM, and Cu 2+ molar final concentration is 100 μmM; (5) The four kinds of high molecular SF spinning solutions obtained in step (4) are treated by electrospinning process under the condition of 30 °C and voltage of 20 kV (receiving distance is 10 cm, and the drum rotating speed is 1500 rpm) to obtain four kinds of electrospinning films SF with piezoelectricity; (6) The electrospinning films obtained in step (5) are placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0042] Example 9 (1) 100 g of silk is put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate is added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water is 1:2:8) is added after drying for dissolution, dialysis, and centrifugation, filtration to obtain SF solution; (2) The SF solution is subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid is dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 10%; (4) Four kinds of metal ion chloride powders of calcium chloride, potassium chloride, magnesium chloride or copper chloride are added to the high molecular solution obtained in step (3), wherein Ca 2+ , K + , Mg 2+ molar final concentration is 20 mM, and Cu 2+ molar final concentration is 100 μmM; (5) The four kinds of high molecular SF spinning solutions obtained in step (4) are treated by electrospinning process under the condition of 30 °C and voltage of 20 kV (receiving distance is 10 cm, and the drum rotating speed is 2000 rpm) to obtain four kinds of electrospinning films SF with piezoelectricity; (6) The electrospinning films obtained in step (5) are placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0043] Example 10 (1) 100 g of silk is put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate is added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water is 1:2:8) is added after drying for dissolution, dialysis, and centrifugation, filtration to obtain SF solution; (2) The SF solution is subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid is dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 10%; (4) To the polymer solution obtained in step (3), calcium chloride or potassium chloride or magnesium chloride or copper chloride powder of four metal ions was added, wherein Ca 2+ , K + , Mg 2+ , and Cu 2+ had a final molar concentration of 20 mM and 100 μmM, respectively; (5) The four kinds of polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30 °C and a voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 3000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0044] Example 11 (1) 100 g of silk was placed in 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, and after drying, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added for dissolution, dialysis, and centrifugation, filtration to obtain SF solution; (2) The SF solution was subjected to freezing treatment, and then vacuum dried to obtain SF solid; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a polymer solution with a mass percentage concentration of 10%; (4) To the polymer solution obtained in step (3), calcium chloride or potassium chloride or magnesium chloride or copper chloride or zinc chloride powder of five metal ions was added, wherein Zn 2+ , Ca 2+ , K + , Mg 2+ , and Cu 2+ had a final molar concentration in the range of 0.01-20 mM, respectively; (5) The five kinds of polymer SF spinning solutions obtained in step (4) were treated by electrospinning process at 30 °C and a voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 2000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40 °C for 10 min, then soaked in methanol for 5 h, dried and rolled into tubes to obtain five kinds of nerve conduits.

[0045] Comparative Example 1 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added after drying for dissolution, dialysis, and centrifugation, filtration to obtain an SF solution; (2) The SF solution was subjected to freezing treatment, and SF solid was obtained after vacuum drying; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 15%; (4) Four kinds of metal ion chloride powders of calcium chloride, potassium chloride, magnesium chloride or copper chloride were added to the high molecular solution obtained in step (3), wherein the molar final concentration of Ca 2+ , K + , Mg 2+ was 25 mM, and the molar final concentration of Cu 2+ was 100 μmM; (5) The four kinds of high molecular SF spinning solutions obtained in step (4) were subjected to electrospinning process treatment under the condition of 30°C and a voltage of 20 kV (the receiving distance was 10 cm, and the drum rotation speed was 2000 rpm) to obtain four kinds of electrospun films SF with piezoelectricity; (6) The electrospun films obtained in step (5) were placed at 40°C for 10 min, then soaked in methanol for 5 h, dried and rolled into tubes to obtain four kinds of nerve conduits.

[0046] Comparative Example 2 (1) 100 g of silk was put into 3 L of deionized water boiled in advance, 20 g of anhydrous sodium carbonate was added for degumming treatment, 240 mL of calcium chloride / anhydrous ethanol / water ternary solution (the molar ratio of calcium chloride, anhydrous ethanol and water was 1:2:8) was added after drying for dissolution, dialysis, and centrifugation, filtration to obtain an SF solution; (2) The SF solution was subjected to freezing treatment, and SF solid was obtained after vacuum drying; (3) 2 g of SF solid was dissolved in dichloromethane to obtain a high molecular solution with a mass percentage concentration of 25%; (4) Four kinds of metal ion chloride powders of calcium chloride, potassium chloride, magnesium chloride or copper chloride were added to the high molecular solution obtained in step (3), wherein the molar final concentration of Ca 2+ , K + , Mg 2+ was 20 mM, and the molar final concentration of Cu 2+ was 100 μmM; (5) The four polymer SF spinning solutions obtained in step (4) are subjected to electrospinning process at 30 ℃ and 20 kV (receiving distance is 10 cm and roller speed is 2000 rpm) to obtain an electrospinned film SF with piezoelectricity. (6) The electrospun membrane obtained in step (5) was left to stand at 40 °C for 10 min, and then soaked in methanol for 5 h and rolled into a tube to obtain the four kinds of nerve conduits.

[0047] Further comparison of the piezoelectric properties of the electrospun membranes in the SF neural scaffolds prepared in Examples 1, 2, and 3 revealed that adjusting the soaking time in methanol significantly affected the piezoelectricity of the electrospun membranes. Soaking for 3 hours resulted in a piezoelectric value that was too low to provide adequate electrical stimulation for the nerve regeneration process; while soaking for 8 hours resulted in a piezoelectric output value that was too high, severely impacting the mechanical properties of the electrospun membrane and reducing its tensile strength. The piezoelectric value measured after 5 hours of soaking provided adequate electrical stimulation for the nerve regeneration process, and the mechanical properties also met the requirements.

[0048] Further comparison of the ultrasonically induced piezoelectric properties of the electrospun SF films prepared in Examples 1-5, Comparative Example 1, and Comparative Example 2 using digital source tables revealed that (since metal ions do not affect the piezoelectricity of the spun film, the spun film tested was represented by one doped with magnesium ions). The measured open-circuit voltage / current values ​​increased with increasing immersion time, from 50.0 ± 50 mV to 600.6 ± 50 mV (see Table 1). These results indicate that the immersion time in the organic solvent has a significant impact on the piezoelectric properties of SF; both excessively long and short immersion times have adverse effects.

[0049] Table 1. Effects of different soaking times on the piezoelectricity and conductivity of SF electrospun films. Further comparison was made of the electrospun membrane SF and CCK8 assay in the four neural scaffolds prepared in Example 11. Figure 4 and Figure 5 It can be found that when the content of certain metal ions is too high, such as when the content of copper ions exceeds 100 μmM, there will be certain toxicity, leading to nerve cell death. Examples 4, 5, 6 and 7 demonstrate that the content of certain metal ions, such as magnesium ions, can promote cell proliferation when the content is below 20 mM, and the content in Example 5 has the best effect on promoting proliferation (see Table 2). However, exceeding 20 mM will also cause cell death. The results show that the content needs to be adjusted according to different ions.

[0050] Table 2. SF / Mg ratios in Examples 2, 4-7 and Comparative Example 1 2+ OD values ​​at 1, 3 and 7 days By further comparing the morphology and mechanical properties of the electrospun SF in the SF nerve scaffolds prepared in Example 8, Example 9, Example 10, Comparative Example 1 and Comparative Example 2, it can be found that adjusting the rotation speed of the spinning machine has a significant effect on the orientation of electrospinning. The rotation speed of Example 8 is 1500 rpm, and the filaments in the spinning film are observed by scanning electron microscopy (SEM) to be adhered to each other, disordered and without orientation effect; the rotation speed of Example 10 is 3000 rpm, which has a certain orientation effect, but the adhesion degree is the highest, and the spinning diameters are also different in size, both of which are not conducive to nerve recovery; the rotation speed of Example 9 is 2000 rpm, and the obtained four kinds of metal ion doped electrospun films are observed by SEM to have a good orientation effect, and the diameters are uniform in size. Figure 1 Comparative Example 9 and Comparative Example 2 also show that the content of SF also affects the stress and strain of the spinning film. Comparative Example 2 shows that too high SF content will also lead to insufficient tensile force. According to statistics, the average diameter of SF / Mg 2+ The average deviation angle of the spinning film is 24.0° ± 0.9° (see Table 3).

[0051] Table 3 Influence of different rotation speeds on SF / Mg 2+ Table of influence of electrospun film deviation angle and the like Figure 1 The SF spinning film morphology and mechanical characterization diagram of Example 9. By scanning electron microscopy, it can be seen that when the rotation speed is 2000 rpm, the spinning film has obvious orientation, the adhesion degree is the lowest, and statistical analysis such as SF / Mg 2+ The average diameter of the spinning film is 0.46 ± 0.07 μm, the average deviation angle is 24.0° ± 0.9° (A, B), the metal ions are evenly dispersed between the spinning films (C). In addition, the strain rate, maximum tensile strength and corresponding Young's modulus of the material in the parallel and vertical directions can be clearly seen, and the electrospun film in the parallel direction exhibits good tensile properties (D, E). By testing the contact angle and infrared of the material, it can be observed that the spinning films of different ions all exhibit good hydrophobicity, and the SF spinning film has a β-sheet conformation transition characteristic, which confirms that the SF spinning film has piezoelectric properties (F, G).

[0052] Figure 2The morphology and structure characterization of SF spinning film prepared in Example 9 are shown in the figure. The X-ray diffraction pattern shows that the β conformation of SF spinning film is significantly improved after organic solvent immersion (A). As can be seen from the butterfly curve and phase diagram, the SF spinning film has good piezoelectricity and is responsive to ultrasonic stimulation (B-H). By testing the ultrasonic effect at different distances and frequencies, it can be found that the piezoelectric value and conductive value of the SF spinning film produced by ultrasonic can perfectly match the electrical stimulation required for nerve regeneration (I-L).

[0053] Figure 3 The ultrasonic release behavior of metal ions in the SF spinning film prepared in Example 9. After testing the calibration curve (A-C), first test the short-term release of three metal ions (Ca 2+ , Mg 2+ , K + ) in the spinning film (D-F), it can be observed that the application of ultrasonic stimulation can significantly stimulate the release behavior of metal ions. By comparing with the long-term release behavior (H-I), the application of ultrasonic stimulation can significantly improve the release rate of metal ions, which proves that the controlled release of metal ions can be realized by ultrasonic stimulation, and the repair efficiency can be improved.

[0054] Figure 4 、 Figure 5 The immunostaining and CCK8 diagram of SF spinning film sample described in Example 11 at the cell level. The live / dead staining diagram of the co-culture of the extract of the spinning film and PC12 cells after 1, 3, 7 days shows that the test results are consistent with the CCK-8 quantitative results, and after the co-culture of the spinning film and PC12 cells, most of the PC12 cells show green staining results (live cells), indicating that the material has good biocompatibility. It can also be proved that the addition of metal ions can significantly improve the proliferation of cells, and the optimal concentration of different ions is different, and too high concentration of some ions will cause cell death, so the concentration needs to be adjusted according to the specific ion.

[0055] Figure 6A simple schematic diagram of the SF spun film of Example 9 to promote cell proliferation and neuron growth at the cellular level. Different colored balls represent different types of metal ions that act on growth factors NGF / BDNF / CDNF (collectively referred to as neurotrophins) in motor neurons and support the restoration of lost neuromuscular interactions. These growth factors mediate their effects through TrkB, which further induces the phosphorylation of other tyrosine residues as docking sites for adapter proteins, initiating further intracellular signaling pathways to promote neuronal differentiation, prevent apoptosis, and improve the survival of motor neurons after a dorsal root avulsion injury. This can protect motor neurons from various injuries, such as copper and zinc, which can regulate the activity of NTs in different ways. In addition, TRPA1 is a non-selective cation channel expressed in sensory neurons and acts as a pain receptor. Some metal ions, such as Ca, Mg, Ba, and Zn, can regulate the activity of the TRPA1 channel to promote the step-by-step regeneration of sensory neurons.

[0056] The above examples are only used to help understand the method and core idea of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the scope of the claims of the present application.

Claims

1. A method for preparing a differential neural repair conduit for sensory / motor neurons based on ultrasound-triggered controllable release of metal ions, characterized in that, Includes the following steps: (1) The silk is placed in pre-boiled deionized water, and anhydrous sodium carbonate is added for degumming to obtain silk fibroin SF. After drying, lithium bromide or calcium chloride, anhydrous ethanol and water ternary solution are added for dissolution and dialysis, and then centrifuged and filtered to obtain S1 solution. (2) After freezing the S1 solution, vacuum drying was performed to obtain solid S2; (3) Dissolve solid S2 in an organic solvent, then add chloride containing metal ions, and stir thoroughly. The metal ions combine with SF based on metal coordination bonds to obtain S3 solution. (4) The S3 solution was treated by electrospinning process, and the relevant parameters were controlled to obtain an oriented electrospinned membrane S4; (5) The electrospun membrane S4 described in step (4) is soaked in an organic solvent, then removed, dried and rolled into a conduit shape to obtain the nerve repair conduit. The organic solvent soaking method enables the nerve repair conduit to respond to different degrees of ultrasonic stimulation to achieve controlled release of metal ions.

2. The method according to claim 1, characterized in that: In step (1), the molar ratio of lithium bromide or calcium chloride, anhydrous ethanol and water in the ternary solution of lithium bromide or calcium chloride, anhydrous ethanol and water is 1:2:

8.

3. The method according to claim 1, characterized in that: The freezing treatment method described in step (2) is liquid nitrogen treatment or placing it in a refrigerator, with a vacuum drying time of 1-4 days.

4. The method according to claim 1, characterized in that: The organic solvent mentioned in step (3) is one or two of chloroform, dichloromethane, hexafluoroisopropanol, and methanol.

5. The method according to claim 1, characterized in that: The metal ion chloride mentioned in step (3) is one of zinc chloride, potassium chloride, magnesium chloride, calcium chloride, and copper chloride, and the concentration of metal ions in the S3 solution is 0.1-20.0 mM.

6. The method according to claim 1, characterized in that: The mass percentage concentration of SF in the S3 solution mentioned in step (3) is 5.0%-20.0%.

7. The method according to claim 1, characterized in that: The electrospinning process described in step (4) is performed at a temperature of 15-50 ℃, a voltage of 5-20 kV, a receiving distance of 5.0-18.0 cm, and a roller speed of 800-3000 rpm.

8. The method according to claim 1, characterized in that: The organic solvent mentioned in step (5) is one or two of methanol, ethanol, and propanol. The soaking time in the organic solvent is 1.0-9.0 h, the drying temperature is 20-60 ℃, and the drying time is 1.0-24.0 h.

9. The method according to claim 1, characterized in that: The different levels of ultrasound stimulation mentioned in step (5) include ultrasound power of 0.5-3.0 W / cm². 2 The ultrasound duration is 0.5-1.0 h, and the ultrasound stimulation distance is 0.5-4.0 cm.

10. A differential neural repair conduit for sensory / motor neurons based on the controllable release of metal ions triggered by ultrasound, prepared by the method according to any one of claims 1-9, characterized in that... The nerve conduit is composed of SF electrospun membrane and metal ions.

Images