Differentiated'inner core and outer sheath 'triboelectric stimulation bionic nerve graft and preparation method thereof

Abstract

The invention relates to a differentiated'inner core and outer sheath 'triboelectric stimulation bionic nerve graft and a preparation method thereof. The nerve graft is composed of an outer sheath layer and an inner core layer. Wherein an outer sheath layer is a calcium ion cross-linked waterborne polyurethane / sodium alginate double-network structure hollow conduit, and an inner core layer is oriented conductive polymer / silk fibroin nanofibers. The nerve graft provided by the invention is provided with the sheath part with an internal and external differential gradient hole structure and the bionic inner core similar to a natural nerve structure, when the nerve graft is implanted into a body, the inner core and the outer sheath show differential degradation speeds and hydrophilic-hydrophobic properties, and can generate electrical stimulation synchronously responding to physiological activities, so that regeneration after nerve injury is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of nerve grafts, and in particular relates to a differentiated "inner core and outer sheath" triboelectric stimulation bionic nerve graft and a preparation method thereof. Background Art

[0002] Peripheral nerve injury often occurs secondary to high-energy mechanical trauma such as sports and accidents. Patients experience varying degrees of sensory and motor dysfunction and muscle paralysis, resulting in a high disability rate that severely impacts their quality of life. Functional repair of peripheral nerve defects is a clinical challenge that urgently needs to be addressed. Currently, surgical treatment is the most common approach for peripheral nerve injury, often involving end-to-end suture or the use of allogeneic, autologous, and tissue-engineered nerve grafts. Anti-immune drugs are often used to suppress the immune response in allogeneic and xenogeneic transplantation, but this carries the risk of infection and tumor formation. Autologous nerve transplantation provides appropriate neurotrophic factors and viable stem cells for regenerating axons. However, autologous transplantation requires the harvesting of donor nerves, which can lead to a range of complications, including impaired nerve function at the donor site, hypertrophic scarring, and painful neuroma formation. Furthermore, these procedures are subject to numerous drawbacks, including limited donor availability and size mismatch. Therefore, the search for suitable alternatives to autologous nerve transplantation is crucial.

[0003] The development and application of artificial nerve grafts has opened up new avenues for repairing peripheral nerve defects. However, current artificial nerve grafts used in clinical practice suffer from insufficient mechanical properties of single materials, lack natural neural biomimetic microstructures, and cannot meet the dynamic microenvironment required for nerve regeneration. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a differentiated "inner core and outer sheath" friction electric stimulation bionic nerve graft and its preparation method. The nerve graft has a sheath with differentiated pore density inside and outside and an inner core similar to a natural nerve. When actually used in the body, the inner core and outer sheath exhibit differentiated degradation rates and hydrophilicity and hydrophobicity, and can also generate electrical stimulation that responds synchronously with physiological activities, which is beneficial to nerve regeneration after injury.

[0005] The present invention provides a differentiated "inner core and outer sheath" triboelectric stimulation bionic nerve graft, which consists of an outer sheath layer and an inner core layer; the outer sheath layer is a hollow conduit with a calcium ion cross-linked aqueous polyurethane / sodium alginate double network structure, and the inner core layer is an oriented conductive polymer / silk fibroin nanofiber.

[0006] Preferably, the inner surface of the outer sheath layer is loose and porous, the outer surface is dense, and the pore size is 10-500 μm.

[0007] Preferably, the inner core layer has a diameter of 10-1000 nm and is oriented along the long axis of the nerve conduit.

[0008] Preferably, the conductive polymer is poly(3,4-ethylenedioxythiophene).

[0009] The present invention also provides a method for preparing a differentiated "inner core and outer sheath" triboelectric stimulation bionic nerve graft, comprising the following steps:

[0010] (1) preparing a waterborne polyurethane (WPU) solution;

[0011] (2) stirring and mixing the WPU solution and the sodium alginate SA solution and then allowing to stand to obtain a sheath spinning solution;

[0012] (3) stirring and mixing the conductive polymer solution and the silk fibroin SF solution and then allowing to stand to obtain a core layer spinning solution;

[0013] (4) Using CaCl2 aqueous solution as a coagulation bath, the sheath spinning solution and the core spinning solution are squeezed into the coagulation bath through a syringe pump and a coaxial needle to obtain a solid fiber;

[0014] (5) The solid fibers are placed vertically on a cold platform, freeze-dried after complete crystallization, and a differentiated “inner core and outer sheath” triboelectric stimulation biomimetic nerve graft is obtained.

[0015] Preferably, the preparation method of the aqueous polyurethane WPU solution in step (1) is as follows:

[0016] Take 5-10g of polycaprolactone diol, dehydrate at 100-120℃ for 1-2.5h, cool to 70-90℃, add 4-5g of isophorone diisocyanate and prepolymerize for 1-3h, after the temperature drops to 50-70℃, add 1-1.5g of dihydroxybutyric acid and 5-15ml of tetrahydrofuran, react for 20-25h, then add 1-1.5g of triethylamine and react for 0.5-1h, after the temperature drops to 15-25℃, add 45-55ml of deionized water, stir at 1000-2000r / min for 1.5-3h, and obtain WPU solution by rotary evaporation in a 45-55℃ water bath.

[0017] Preferably, the mass of the WPU solution weighed in step (2) is 5-10 g, and the mass fraction of the SA solution added is 1%-3%.

[0018] Preferably, in step (3), the mass of SF is weighed to be 0.4-0.9 g, the mass of the conductive polymer added is 0.1-0.6 g, and the two are dissolved in 10-30 ml of deionized water.

[0019] Preferably, the mass fraction of the conductive polymer solution in step (3) is 1%-6%, and the mass fraction of the silk fibroin SF solution is 99%-94%.

[0020] Preferably, the spinning speed of the core layer spinning solution in step (4) is 45-55 ml / h, and the spinning speed of the sheath layer spinning solution is 30-60 ml / h.

[0021] Preferably, the mass fraction of the CaCl2 aqueous solution in step (4) is 1%-3%.

[0022] Preferably, the volume of the injection pump in step (4) is 15-25 ml.

[0023] Preferably, the diameter of the needle in step (4) is 2-4 mm.

[0024] Preferably, the freeze-drying time in step (5) is 20-25 hours.

[0025] The waterborne polyurethane of the present invention is based on the design of the polyurethane molecular structure. Hydrophilic ionic groups are introduced into the main chain or side chain to disperse the polyurethane in the aqueous solution in the form of an emulsion. The hydrophilic groups can also undergo rapid ion complexation with divalent metal ions. Sodium alginate is a common marine polysaccharide with excellent biocompatibility and degradability. The repeating units in its molecular chain can react with calcium ions (Ca 2+ ) complexation to form a gel network structure. By modifying waterborne polyurethane with sodium alginate, the 2+ The rapid complexing ability of silk can realize wet spinning. Silk fibroin is derived from the degumming process of silk. It is a high-molecular-weight polypeptide composed of various amino acids. It has good biocompatibility, biodegradability, rich regulatory properties, etc., and can provide a good microenvironment support for nerve regeneration. Conductive polymers can stably conduct electricity under physiological conditions and have low toxicity, which is conducive to the adhesion, growth and differentiation of nerve cells. The directional freezing method uses a unidirectional low-temperature source to provide a freezing gradient, uses ice crystals as a pore-forming template, and is a simple and efficient template method to control the directional movement of frozen elements.

[0026] Beneficial effects

[0027] (1) The nerve graft of the present invention has a sheath with differentiated pore densities inside and outside and an inner core similar to a natural nerve. When actually used in vivo, the inner core and outer sheath exhibit differentiated degradation rates and hydrophilicity and hydrophobicity. It can also generate electrical stimulation that responds synchronously with physiological activities, which is beneficial to the regeneration of nerves after injury.

[0028] (2) The outer surface of the nerve graft tube of the present invention is dense, the inner surface is loose and porous, and the core has an oriented multi-channel structure, which is conducive to cell adhesion, proliferation and directional migration. In addition, it has excellent charge transfer, storage and triboelectric capabilities, which is conducive to electrical stimulation of tissue regeneration.

[0029] (3) The present invention uses a conductive polymer as the inner core, which can not only ensure the conductivity of the nerve graft, but also have excellent triboelectric capacity when actually implanted in the body, which is conducive to electrical stimulation to repair tissue regeneration.

[0030] (4) The present invention adopts directional freeze-drying technology to prepare the inner core, which is similar to the growth direction of natural nerves and is conducive to cell adhesion, proliferation and directional migration.

[0031] (5) The present invention utilizes a coaxial wet spinning process to prepare a nerve graft, which has a simple and controllable process and can customize the size of the nerve graft. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a SEM image of the cross section of the nerve graft of the present invention.

[0033] Figure 2 Schematic diagram of the preparation process of the nerve graft of the present invention.

[0034] Figure 3 1 is a SEM image of the inner and outer walls of the outer sheath of the nerve graft of the present invention.

[0035] Figure 4 This is a SEM image of the inner core of the nerve graft of the present invention.

[0036] Figure 5 The graph shows the tribovoltage test results of the nerve graft of the present invention.

[0037] Figure 6 The figure shows the tribocurrent test results of the nerve graft of the present invention. DETAILED DESCRIPTION

[0038] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0039] Example 1

[0040] This embodiment provides a method for preparing a differentiated "inner core and outer sheath" triboelectric stimulation bionic nerve graft, comprising the following steps:

[0041] (1) Take 10g of polycaprolactone diol, remove water at 110℃ for 2h, cool to 80℃, add 4.45g of isophorone diisocyanate and prepolymerize for 2h, wait until the temperature drops to 60℃, add 1.34g of dihydroxybutyric acid and 10ml of tetrahydrofuran, react for 22h, then add 1.01g of triethylamine and neutralize for 0.5h, wait until the temperature drops to 20℃, add 50ml of deionized water, stir at 1500r / min for 2h, and obtain WPU solution by rotary evaporation in a 50℃ water bath.

[0042] (2) 10 g of WPU solution and 0.2 g of sodium alginate SA solution were stirred and mixed, and then allowed to stand to obtain a sheath spinning solution.

[0043] (3) 0.2 g of conductive polymer and 0.8 g of silk fibroin SF were mixed, added into 20 ml of deionized water, stirred and mixed, and then allowed to stand to obtain a core layer spinning solution.

[0044] (4) Using a 2% CaCl2 aqueous solution as a coagulation bath, a 25ml syringe and a 3mm diameter needle were selected as the spinning outlet, and the core layer spinning solution and the sheath layer spinning solution were extruded at a speed of 50ml / h and 30ml / h, respectively. The solid fibers were squeezed into the coagulation bath through a coaxial needle to obtain solid fibers.

[0045] (5) The solid fibers were placed vertically on a cold platform and freeze-dried for 24 h after complete crystallization to obtain a differentiated “inner core and outer sheath” triboelectric stimulation biomimetic nerve graft, as shown in the SEM image. Figure 1 shown.

[0046] Figure 3 The results showed that the inner and outer walls of the prepared nerve graft sheath had differentiated pore structures.

[0047] Figure 4 The results showed that the inner core of the prepared nerve graft had an oriented structure.

[0048] Figure 5 The results showed that the inner core and outer sheath of the prepared nerve graft could generate friction voltage.

[0049] Figure 6 The results showed that the inner core and outer sheath of the prepared nerve graft can generate friction current.

Claims

1. A differentiated "inner core, outer sheath" triboelectric stimulation biomimetic nerve graft, characterized by: The nerve graft consists of an outer sheath layer and an inner core layer; the outer sheath layer is a hollow conduit with a calcium ion cross-linked aqueous polyurethane / sodium alginate double network structure, and the inner core layer is an oriented conductive polymer / silk fibroin nanofiber.

2. The nerve graft according to claim 1, wherein: The inner surface of the outer sheath layer is loose and porous, the outer surface is dense, and the pore size is 10-500 μm.

3. The nerve graft according to claim 1, wherein: The inner core layer has a diameter of 10-1000 nm and is oriented along the long axis of the nerve conduit.

4. The nerve graft according to claim 1, wherein: The conductive polymer is poly (3,4-ethylenedioxythiophene).

5. A method for preparing a differentiated "inner core and outer sheath" triboelectric stimulation biomimetic nerve graft, comprising the following steps: (1) preparing a waterborne polyurethane (WPU) solution; (2) stirring and mixing the WPU solution and the sodium alginate SA solution and then allowing to stand to obtain a sheath spinning solution; (3) dissolving the conductive polymer and silk fibroin SF in deionized water, stirring and mixing, and then allowing to stand to obtain a core layer spinning solution; (4) Using CaCl2 aqueous solution as a coagulation bath, the sheath spinning solution and the core spinning solution are squeezed into the coagulation bath through a syringe pump and a coaxial needle to obtain a solid fiber; (5) The solid fibers are placed vertically on a cold platform, freeze-dried after complete crystallization, and a differentiated "inner core and outer sheath" triboelectric stimulation bionic nerve graft is obtained.

6. The preparation method according to claim 5, characterized in that: The preparation method of the aqueous polyurethane WPU solution in the step (1) is as follows: Take 5-10g of polycaprolactone diol, dehydrate at 100-120℃ for 1-2.5h, cool to 70-90℃, add 4-5g of isophorone diisocyanate and prepolymerize for 1-3h, after the temperature drops to 50-70℃, add 1-1.5g of dihydroxybutyric acid and 5-15ml of tetrahydrofuran, react for 20-25h, then add 1-1.5g of triethylamine and react for 0.5-1h, after the temperature drops to 15-25℃, add 45-55ml of deionized water, stir at 1000-2000r / min for 1.5-3h, and obtain WPU solution by rotary evaporation in a 45-55℃ water bath.

7. The preparation method according to claim 5, characterized in that: In the step (2), 5-15 g of the synthesized WPU solution is weighed, and the SA solution is added so that its mass fraction in the mixed solution accounts for 1%-3%.

8. The preparation method according to claim 5, characterized in that: In the step (3), 0.1-0.6 g of the conductive polymer and 0.4-0.9 g of the SF solution are weighed and dissolved in 10-30 ml of deionized water to obtain a core layer spinning solution.

9. The preparation method according to claim 5, characterized in that: The spinning speed of the core layer spinning solution in the step (4) is 45-55 ml / h, and the spinning speed of the sheath layer spinning solution is 30-60 ml / h.

10. The preparation method according to claim 5, characterized in that: The freeze-drying time in step (5) is 20-25 hours.

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