WO2022077878A1 - Silk protein nerve conduit and preparation method therefor - Google Patents

Silk protein nerve conduit and preparation method therefor Download PDF

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Publication number
WO2022077878A1
WO2022077878A1 PCT/CN2021/086167 CN2021086167W WO2022077878A1 WO 2022077878 A1 WO2022077878 A1 WO 2022077878A1 CN 2021086167 W CN2021086167 W CN 2021086167W WO 2022077878 A1 WO2022077878 A1 WO 2022077878A1
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silk protein
conduit
catheter
silk
nerve
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PCT/CN2021/086167
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French (fr)
Chinese (zh)
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吕强
路青青
张锋
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/02—Methods for coating medical devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • the invention belongs to the technical field of biomedical materials, and in particular relates to a silk protein nerve conduit and a preparation method thereof.
  • Peripheral nerve injury is a common clinical condition, and autologous transplantation is often used to bridge peripheral nerve defects.
  • limited availability and donor site morbidity still exist, so to overcome this limitation, researchers developed biomaterial-based nerve conduits to assist peripheral nerve regeneration. Artificial conduits have been shown to have the ability to fill nerve gaps, synthetic nerve conduits are expected to replace autografts, and improvements in biomaterials hold great potential for promoting nerve regeneration and functional recovery. Therefore, the developed nerve conduit not only has good biological properties, but also should provide a better microenvironment for the regeneration of nerve tissue.
  • Silk protein has been widely used in the repair and regeneration of different tissues due to its excellent biocompatibility, degradability, and low inflammatory response, and good early results have also been obtained in the field of neuroreparation.
  • how to use silk protein as a matrix ?
  • New preparation methods need to be considered to obtain better orientation induction. Signaling of silk protein conduits.
  • a composite nerve conduit with excellent mechanical properties on the outside and meeting application requirements, and an oriented silk protein sponge on the inside to provide orientation-inducing signals can be prepared to achieve better repair effect.
  • the key of the present invention is not only to solve the problem of stable acquisition of the insolubility of the silk protein braided tube and the silk protein film in the aqueous environment, but also to solve the technical problem of the efficient introduction of the oriented sponge into the catheter.
  • the technical problem to be solved by the present invention is to provide a silk protein nerve conduit with an orientation-inducing signal and a preparation method thereof.
  • the invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
  • the present invention also provides a preparation method of silk protein nerve conduit, comprising:
  • the fibroin fiber is 80-100 denier degummed silk; the braiding angle is 45°-60°; the braiding speed is 30-60 r/min; the inner diameter of the catheter base is 0.5-15 mm.
  • the concentration of silk protein in the silk protein solution is 2-10 wt%; the silk protein comprises silk protein nanofibers; the mass of the silk protein nanofibers is 0%-40% of the mass of the silk protein.
  • the silk protein nanofibers have a diameter of 10-20 nm and a length of 1-2 ⁇ m.
  • described S2) is specifically:
  • the catheter substrate is soaked in the silk protein solution and evaporated at 10°C to 90°C to obtain the catheter part.
  • the silk protein nanofiber gel is prepared according to the following steps:
  • the second silk protein nanofiber aqueous solution is diluted with water to a third silk protein nanofiber aqueous solution with a concentration of 0.2-4wt%, and then sealed and incubated at 50°C to 70°C to obtain a silk protein nanofiber gel.
  • the step S3) is specifically:
  • the silk protein nanofiber gel is induced by an electric field to obtain a silk protein nanofiber gel with an oriented structure
  • the catheter part is inserted into the silk protein nanofiber gel with an oriented structure along the orientation direction, so that the catheter part is filled, and after being taken out, freeze-dried to obtain the silk protein nerve catheter;
  • the catheter part is immersed in the silk protein nanofiber gel along the direction parallel to the electrode, and then subjected to electric field treatment, and after being taken out, freeze-dried to obtain the silk protein nerve catheter.
  • the electric field strengths of the electric field induction and electric field treatment are each independently 10-120V; and the time is each independently 5-60min.
  • the freeze-drying is specifically: firstly freeze at -20°C to -50°C for 1 to 12 hours, and then freeze-dry at -2°C to -90°C for 24 to 72 hours.
  • the invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein
  • the fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
  • a silk protein nanofiber sponge with an oriented structure is arranged inside the catheter, and has an oriented structure consistent with the direction of the nerve axon, which can provide an orientation inducing signal, promote the migration and increase of nerve cells, and can also provide an orientation-inducing signal. It guides the regeneration and extension of nerve fibers and significantly improves the function of nerve conduits; therefore, the present invention solves the problems of mechanical properties and orientation induction through the combination of silk protein tubes and silk protein orientation sponges.
  • Fig. 1 is the scanning electron microscope picture of the braided catheter obtained in the step (1) of the embodiment of the present invention 1;
  • Fig. 3 is the scanning electron microscope picture of the silk protein nerve conduit obtained in the embodiment of the present invention 2;
  • Fig. 4 is the scanning electron microscope image of the oriented silk protein sponge inside the silk protein nerve conduit obtained in Example 3 of the present invention.
  • Figure 5 is an electrophysiological waveform diagram of the regenerated nerve and autologous transplantation after the silk protein nerve conduit obtained in Example 3 of the present invention was transplanted into the rat sciatic nerve defect.
  • the invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
  • the invention improves the mechanical properties of the catheter by compounding the silk protein film on the surface of the catheter matrix formed by weaving the silk protein fibers; the silk protein nanofiber sponge with the orientation structure is arranged inside the catheter, and the orientation structure is consistent with the direction of the nerve axon. It can provide orientation-inducing signals, promote the migration and proliferation of nerve cells, and also guide the regeneration and extension of nerve fibers, significantly improving the function of nerve conduits; therefore, the present invention solves mechanical properties and orientation through the combination of silk protein tubes and silk protein orientation sponges. induced problems.
  • the present invention also provides a method for preparing the above silk protein nerve conduit, comprising: S1) weaving silk protein fibers to obtain a conduit matrix; S2) soaking the conduit matrix in a silk protein solution to obtain a conduit portion; S3) The silk protein nanofiber gel with oriented structure is combined with the conduit part, and after freeze-drying, the silk protein nerve conduit is obtained.
  • the present invention does not have any special restrictions on the sources of all raw materials, which can be commercially available.
  • the fibroin fiber is braided to obtain a catheter matrix;
  • the fibroin fiber is preferably silk, more preferably degummed silk, and more preferably degummed silk of 80-100 deniers;
  • the braiding angle of the braiding is preferably 45° ⁇ 60°;
  • the speed of the weaving is preferably 30-60r/min, more preferably 40-50r/min, and more preferably 45r/min;
  • the gear ratio during the weaving is preferably (60-100): (20-60), more preferably It is preferably (70-90):(30-50), more preferably 80:40;
  • the woven skeleton structure is preferably a knitted structure or a woven structure;
  • the weaving is preferably performed on a smooth metal mold;
  • the diameter of the mold is preferably 0.5 to 15 mm, so that the inner diameter of the resulting catheter base is preferably 0.5 to 15 mm.
  • the catheter substrate is soaked in the silk protein solution, preferably the catheter substrate with the mold is soaked in the silk protein solution; the concentration of the silk protein in the silk protein solution is preferably 2-10wt%; In the embodiments, the concentration of the silk protein solution is preferably 2wt%; in some embodiments provided by the present invention, the concentration of the silk protein solution is preferably 5wt%; in other embodiments provided by the present invention, the The concentration of the silk protein solution is preferably 10wt%; the silk protein in the silk protein solution includes silk protein nanofibers; the quality of the silk protein nanofibers is preferably 0% to 40% of the total mass of the silk protein, more preferably 0.5% to 30% of the total mass of silk protein, preferably 0.5% to 10% of the total mass of silk protein, most preferably 0.8% to 4% of the total mass of silk protein; use silk protein nanofibers to induce the conformational transition of ordinary silk protein
  • the diameter of the silk protein nanofibers is preferably 10-20 nm; the length of the silk protein nanofibers is preferably
  • the silk protein nanofiber gel with an orientation structure is combined with the catheter part; in the present invention, the combination can be performed according to the following method: the silk protein nanofiber gel is induced by an electric field to obtain a silk protein nanofiber gel with an orientation structure. Then insert the catheter part into the silk protein nanofiber gel with the orientation structure along the orientation direction, so that it fills the catheter part; wherein, the silk protein nanofiber gel is preferably carried out according to the following steps: the silk protein nanofiber The aqueous solution is concentrated at 40°C to 60°C to a first silk protein nanofiber aqueous solution with a concentration of 6-12wt%; the first silk protein nanofiber aqueous solution is concentrated at 20°C to 35°C to a concentration of 18-24wt% of the second silk protein Nanofiber aqueous solution; dilute the second silk protein nanofiber aqueous solution with water to a third silk protein nanofiber aqueous solution with a concentration of 0.2-4wt%, and then seal and incubate at 50°C to 70°
  • the catheter part is immersed in the silk protein nanofiber gel along the direction parallel to the electrode, and then electric field treatment is performed; the silk protein nanofiber gel is as described above, and will not be repeated here;
  • the intensity of the electric field treatment is preferably 10-120V; the time of the electric field treatment is preferably 5-60min; the electric field treatment makes the nanofibers inside the conduit move directionally, and forms an orientation gel in the portion near the positive electrode inside the conduit, which occupies
  • the volume is about 1/3, and it is preferable to repeat the above steps of immersion and electric field treatment until the silk protein nanofiber gel with oriented structure fills more than 90% of the catheter part.
  • freeze-drying is performed to obtain a silk protein nerve catheter; the freeze-drying is preferably specifically: firstly freeze at -20°C to -50°C for 1 to 12 hours, and then freeze at -20°C to -50°C for 1 to 12 hours. -2°C ⁇ -90°C freeze drying for 24 ⁇ 72h.
  • the gel is transformed into a porous sponge with an oriented structure, and finally a composite nerve conduit with a conduit on the outside and an oriented sponge on the inside is obtained.
  • the silk protein nerve conduit prepared by the invention does not add any toxic substances, all its components are natural proteins, and have good compatibility; and by regulating the conformational transition rate of the silk protein, the water-insoluble silk protein fiber can be directly and stably obtained in an aqueous solution.
  • the catheter part of the film composite improves the mechanical properties of the catheter and makes it meet the needs of clinical applications.
  • the interior of the catheter is an oriented silk protein nanofiber sponge, which has an oriented structure consistent with the direction of the nerve axon, which can provide orientation-inducing signals and promote neural
  • the migration and proliferation of cells can also guide the regeneration and extension of nerve fibers, and significantly improve the function of nerve conduits; through the combination of silk protein tubes and silk protein orientation sponges, the problems of mechanical properties and orientation induction are solved; All are based on the basic physical treatment process, there is no chemical reaction and harsh preparation conditions, which is conducive to the industrial transformation and functional enrichment of the technology. Although it is a mild physical control, its related parameters and key steps of process integration are all based on silk protein.
  • the silk protein nanofibers used in the examples have a diameter of 10 nm and a length of 1 ⁇ m;
  • the preparation method of the silk protein nanofiber gel is as follows: the silk protein nanofibers (10-20 nm in diameter, 1-2 ⁇ m in length) are concentrated into an aqueous solution at 40° C. to 60° C. to a first silk with a concentration of 6-12 wt %.
  • Protein nanofiber aqueous solution concentrate the first silk protein nanofiber aqueous solution at 20°C to 35°C to a second silk protein nanofiber aqueous solution with a concentration of 18-24 wt %; dilute the second silk protein nanofiber aqueous solution with water to the concentration A 0.2-4wt% aqueous solution of the third silk protein nanofibers, and then sealed and incubated at 50°C to 70°C to obtain a silk protein nanofiber gel;
  • the silk protein in the described embodiment can be prepared according to Reversible Hydrogel-Solution System of Silk with High Beta-Sheet Content (Biomacromolecules 2014, 15, 3044-3051).
  • the braided catheter obtained in step (1) is analyzed with a scanning electron microscope, and its scanning electron microscope image is obtained, as shown in FIG. 1 .
  • the fibroin fiber-fibroin membrane composite conduit obtained in step (2) is analyzed by scanning electron microscope, and its scanning electron microscope image is obtained, as shown in FIG. 2 .
  • the catheter was placed in a direction parallel to the electrodes and immersed in 4% silk protein nanofiber gel, and then the gel was subjected to electric field treatment, the electric field strength was 100V, and the treatment time was 30min, so as to make the nanofibers inside the catheter move directionally , and the oriented gel is formed in the part near the positive electrode inside the catheter, which occupies about 1/3 of the volume, and then continues to be filled with gel in the part of the catheter without oriented gel, and the electric field treatment process is repeated until more than 90% of the space is filled with gel.
  • the orientation gel occupies, the catheter is removed, and the composite catheter is obtained. Put it in a -20°C refrigerator overnight, and then put it in a freeze dryer at -50°C for 48 hours to freeze-dry it out to convert the gel into an orientation sponge, and finally obtain a silk protein nerve conduit with an orientation signal.
  • the silk protein nerve conduit obtained in Example 2 was analyzed by scanning electron microscope, and its scanning electron microscope image was obtained, as shown in FIG. 3 .
  • the silk protein nerve conduit obtained in Example 3 was analyzed with a scanning electron microscope, and a scanning electron microscope image of the internally oriented silk protein sponge was obtained, as shown in FIG. 4 .
  • Figure 5 shows the electrophysiological waveforms of regenerated nerves and autologous transplantation after the silk protein nerve conduit obtained in Example 3 was transplanted into the rat sciatic nerve defect. Electrical signaling was detected 12 weeks after nerve conduit transplantation into the rat defect.
  • the present invention discloses an oriented silk fibroin nerve conduit and a preparation method thereof.
  • a nerve conduit which is respectively composed of a silk-based conduit and an oriented silk protein nanofiber scaffold is designed.
  • the silk protein conduit reinforced by silk woven fibers is filled with silk protein gel with an orientation structure, and the gel is transformed into an orientation sponge by freeze-drying technology to obtain a nerve conduit with orientation-inducing signals.
  • the preparation process of the invention has mild conditions, and innovatively combines different silk protein preparation technologies to obtain a nerve conduit with orientation signals and mechanical properties that meet application requirements.

Abstract

Provided in the present invention is a silk protein nerve conduit, comprising a conduit portion and a filling sponge filled within the conduit portion. The conduit portion comprises a conduit base and a silk protein thin film compounded on the surface of the conduit base. The conduit base is formed by means of weaving silk protein fibers. The filling sponge is formed by means of a silk protein nanofiber gel with an oriented structure. Compared with the prior art, the silk protein nerve conduit of the present invention has an orientation structure consistent with the neuro-axial direction by means of arranging the silk protein nanofiber sponge with the oriented structure inside the conduit, can provide an orientation-inducing signal, promote the migration and proliferation of nerve cells, can also guide the regeneration and extension of nerve fibers and significantly improve the function of the neural conduit. Therefore, the problems of mechanical performance and orientation induction are solved in the present invention by means of the combination of a silk protein tube and the silk protein oriented sponge.

Description

一种丝蛋白神经导管及其制备方法A kind of silk protein nerve conduit and preparation method thereof
本申请要求于2020年10月15日提交中国专利局、申请号为202011102746.5、发明名称为“一种丝蛋白神经导管及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application filed on October 15, 2020 with the application number 202011102746.5 and titled "A silk protein nerve conduit and its preparation method", the entire contents of which are incorporated by reference in in this application.
技术领域technical field
本发明属于生物医用材料技术领域，尤其涉及一种丝蛋白神经导管及其制备方法。The invention belongs to the technical field of biomedical materials, and in particular relates to a silk protein nerve conduit and a preparation method thereof.
背景技术Background technique
周围神经损伤是临床上常见的的病症，自体移植常用于周围神经缺损的桥接，然而有限的可用性和供体部位的发病率仍然存在，因此为了克服这一限制，研究人员通过开发生物材料为基础的神经导管来辅助周围神经再生。人工导管已被证明有能力填补神经间隙，合成的神经导管有望替代自体移植物，生物材料的改进在促进神经再生和功能恢复方面具有很大的潜力。因此，所研制的神经导管不仅具有良好的生物学性能，还应该为神经组织的再生提供更好的微环境。Peripheral nerve injury is a common clinical condition, and autologous transplantation is often used to bridge peripheral nerve defects. However, limited availability and donor site morbidity still exist, so to overcome this limitation, researchers developed biomaterial-based nerve conduits to assist peripheral nerve regeneration. Artificial conduits have been shown to have the ability to fill nerve gaps, synthetic nerve conduits are expected to replace autografts, and improvements in biomaterials hold great potential for promoting nerve regeneration and functional recovery. Therefore, the developed nerve conduit not only has good biological properties, but also should provide a better microenvironment for the regeneration of nerve tissue.
目前已经有多种不同材料制备的神经导管，并在动物实验和临床研究中取得相对良好的效果，然而同自体的神经导管相比，其性能仍然存在较大差距。如何根据仿生理念，将不同诱导信号引入神经导管的设计，是进一步优化神经导管功能，改善外周神经损伤修复的有效途径。At present, there are many nerve conduits made of different materials, and relatively good results have been achieved in animal experiments and clinical studies. However, compared with autologous nerve conduits, there is still a big gap in their performance. How to introduce different induction signals into the design of the nerve conduit according to the bionic concept is an effective way to further optimize the function of the nerve conduit and improve the repair of peripheral nerve damage.
丝蛋白由于其优异的生物相容性，可降解性以及低的炎症反应，被广泛应用于不同组织的修复和再生，在神经修复领域也获得良好的前期结果，然而，如何以丝蛋白为基质，实现不同诱导信号的主动引入仍然存在较大技术障碍。目前研究者主要通过电纺丝技术以及定向冷冻技术实现取向信号的引入，其同神经导管的有效结合以及技术的稳定性方面存在较大问题，需要考虑新的制备方法来获得具有更好取向诱导信号的丝蛋白导管。Silk protein has been widely used in the repair and regeneration of different tissues due to its excellent biocompatibility, degradability, and low inflammatory response, and good early results have also been obtained in the field of neuroreparation. However, how to use silk protein as a matrix? However, there are still large technical obstacles to realize the active introduction of different inducing signals. At present, researchers mainly use electrospinning technology and directional freezing technology to achieve the introduction of orientation signals. There are major problems in the effective combination with nerve conduits and the stability of the technology. New preparation methods need to be considered to obtain better orientation induction. Signaling of silk protein conduits.
在我们团队的研究中，通过将丝蛋白编织技术和丝蛋白成膜技术的结合，成功获得力学性能良好的复合导管，并将其应用于小血管的替代，然而由于丝蛋白溶液的不稳定性，如何稳定获得不溶于水的丝蛋白纤维-薄膜复合导管仍需要在理解丝蛋白机制的基础上进行技术的改进和突破；另外我们通过电场诱 导丝蛋白纳米纤维定向迁移，成功获得具有取向结构的丝蛋白凝胶或海绵，并将其成功应用于创面修复，以及神经细胞的诱导迁移，但上述凝胶或者海绵力学性能差，难以满足临床应用要求，因此本发明基于团队对丝蛋白的深刻理解，希望通过不同技术的结合和优化，制备外部为力学性能优异、满足应用要求，内部为取向丝蛋白海绵，提供取向诱导信号的复合神经导管，以获得更好的修复效果。本发明的关键不仅要解决丝蛋白编织管和丝蛋白薄膜在水相环境中不溶性的稳定获得问题，同时要解决取向海绵高效引入导管的技术问题。In the research of our team, by combining silk protein weaving technology and silk protein film forming technology, a composite catheter with good mechanical properties was successfully obtained and applied to the replacement of small blood vessels. However, due to the instability of silk protein solution , how to stably obtain water-insoluble silk protein fiber-film composite catheters still requires technical improvements and breakthroughs on the basis of understanding the mechanism of silk protein; in addition, we successfully obtained the directional structure of silk protein nanofibers through electric field-induced directional migration. Silk protein gel or sponge has been successfully applied to wound repair and induced migration of nerve cells. However, the above-mentioned gel or sponge has poor mechanical properties and is difficult to meet the requirements of clinical application. Therefore, the present invention is based on the team's deep understanding of silk protein. It is hoped that through the combination and optimization of different technologies, a composite nerve conduit with excellent mechanical properties on the outside and meeting application requirements, and an oriented silk protein sponge on the inside to provide orientation-inducing signals can be prepared to achieve better repair effect. The key of the present invention is not only to solve the problem of stable acquisition of the insolubility of the silk protein braided tube and the silk protein film in the aqueous environment, but also to solve the technical problem of the efficient introduction of the oriented sponge into the catheter.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明要解决的技术问题在于提供一种具有取向诱导信号的丝蛋白神经导管及其制备方法。In view of this, the technical problem to be solved by the present invention is to provide a silk protein nerve conduit with an orientation-inducing signal and a preparation method thereof.
本发明提供了一种丝蛋白神经导管，包括：导管部分与填充在导管部分内的填充海绵；所述导管部分包括导管基体与复合在导管基体表面的丝蛋白薄膜；所述导管基体由丝蛋白纤维编织形成；所述填充海绵由具有取向结构的丝蛋白纳米纤维凝胶形成。The invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
本发明还提供了一种丝蛋白神经导管的制备方法，包括：The present invention also provides a preparation method of silk protein nerve conduit, comprising:
S1)将丝蛋白纤维进行编织得到导管基体；S1) weaving silk protein fibers to obtain a catheter matrix;
S2)将所述导管基体浸泡在丝蛋白溶液中，得到导管部分；S2) soaking the catheter matrix in the silk protein solution to obtain the catheter part;
S3)将具有取向结构的丝蛋白纳米纤维凝胶与导管部分结合，冷冻干燥后，得到丝蛋白神经导管。S3) combining the silk protein nanofiber gel with an oriented structure with the catheter part, and after freeze-drying, the silk protein nerve catheter is obtained.
优选的，所述丝蛋白纤维为80～100旦脱胶蚕丝；所述编织的编织角为45°～60°；编织速度为30～60r/min；所述导管基体的内径为0.5～15mm。Preferably, the fibroin fiber is 80-100 denier degummed silk; the braiding angle is 45°-60°; the braiding speed is 30-60 r/min; the inner diameter of the catheter base is 0.5-15 mm.
优选的，所述丝蛋白溶液中丝蛋白的浓度为2～10wt％；所述丝蛋白包括丝蛋白纳米纤维；所述丝蛋白纳米纤维的质量为丝蛋白质量的0％～40％。Preferably, the concentration of silk protein in the silk protein solution is 2-10 wt%; the silk protein comprises silk protein nanofibers; the mass of the silk protein nanofibers is 0%-40% of the mass of the silk protein.
优选的，所述丝蛋白纳米纤维的直径为10～20nm；长度为1～2μm。Preferably, the silk protein nanofibers have a diameter of 10-20 nm and a length of 1-2 μm.
优选的，所述S2)具体为：Preferably, described S2) is specifically:
将所述导管基体浸泡在丝蛋白溶液中，在10℃～90℃蒸发，得到导管部分。The catheter substrate is soaked in the silk protein solution and evaporated at 10°C to 90°C to obtain the catheter part.
优选的，所述丝蛋白纳米纤维凝胶按照以下步骤进行制备：Preferably, the silk protein nanofiber gel is prepared according to the following steps:
将丝蛋白纳米纤维水溶液40℃～60℃浓缩至浓度为6～12wt％的第一丝 蛋白纳米纤维水溶液；Concentrating the silk protein nanofiber aqueous solution at 40°C to 60°C to a first silk protein nanofiber aqueous solution with a concentration of 6 to 12wt%;
将第一丝蛋白纳米纤维水溶液在20℃～35℃浓缩至浓度为18～24wt％的第二丝蛋白纳米纤维水溶液；Concentrating the first silk protein nanofiber aqueous solution at 20°C to 35°C to a second silk protein nanofiber aqueous solution with a concentration of 18 to 24 wt %;
将所述第二丝蛋白纳米纤维水溶液加水稀释至浓度为0.2～4wt％的第三丝蛋白纳米纤维水溶液，然后在50℃～70℃密封孵育，得到丝蛋白纳米纤维凝胶。The second silk protein nanofiber aqueous solution is diluted with water to a third silk protein nanofiber aqueous solution with a concentration of 0.2-4wt%, and then sealed and incubated at 50°C to 70°C to obtain a silk protein nanofiber gel.
优选的，所述步骤S3)具体为：Preferably, the step S3) is specifically:
将丝蛋白纳米纤维凝胶利用电场诱导，得到具有取向结构的丝蛋白纳米纤维凝胶；The silk protein nanofiber gel is induced by an electric field to obtain a silk protein nanofiber gel with an oriented structure;
然后将导管部分沿着取向方向插进具有取向结构的丝蛋白纳米纤维凝胶中，使其充满导管部分，取出后，冷冻干燥，得到丝蛋白神经导管；Then the catheter part is inserted into the silk protein nanofiber gel with an oriented structure along the orientation direction, so that the catheter part is filled, and after being taken out, freeze-dried to obtain the silk protein nerve catheter;
或者：将导管部分沿着平行于电极的方向浸入丝蛋白纳米纤维凝胶中，然后进行电场处理，取出后，冷冻干燥，得到丝蛋白神经导管。Or: the catheter part is immersed in the silk protein nanofiber gel along the direction parallel to the electrode, and then subjected to electric field treatment, and after being taken out, freeze-dried to obtain the silk protein nerve catheter.
优选的，所述电场诱导与电场处理的电场强度各自独立地为10～120V；时间各自独立地为5～60min。Preferably, the electric field strengths of the electric field induction and electric field treatment are each independently 10-120V; and the time is each independently 5-60min.
优选的，所述冷冻干燥具体为：先在-20℃～-50℃冷冻1～12h后，然后在-2℃～-90℃冷冻干燥24～72h。Preferably, the freeze-drying is specifically: firstly freeze at -20°C to -50°C for 1 to 12 hours, and then freeze-dry at -2°C to -90°C for 24 to 72 hours.
本发明提供了一种丝蛋白神经导管，包括：导管部分与填充在导管部分内的填充海绵；所述导管部分包括导管基体与复合在导管基体表面的丝蛋白薄膜；所述导管基体由丝蛋白纤维编织形成；所述填充海绵由具有取向结构的丝蛋白纳米纤维凝胶形成。与现有技术相比，本发明通过导管内部设置有具有取向结构的丝蛋白纳米纤维海绵，具有与神经轴突方向一致的取向结构，能够提供取向诱导信号，促进神经细胞的迁移增值，也能指导神经纤维的再生和延伸，显著提高神经导管的功能；因此本发明通过丝蛋白管和丝蛋白取向海绵的结合，解决力学性能和取向诱导的问题。The invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure. Compared with the prior art, in the present invention, a silk protein nanofiber sponge with an oriented structure is arranged inside the catheter, and has an oriented structure consistent with the direction of the nerve axon, which can provide an orientation inducing signal, promote the migration and increase of nerve cells, and can also provide an orientation-inducing signal. It guides the regeneration and extension of nerve fibers and significantly improves the function of nerve conduits; therefore, the present invention solves the problems of mechanical properties and orientation induction through the combination of silk protein tubes and silk protein orientation sponges.
附图说明Description of drawings
图1为本发明实施例1步骤(1)中得到的编织的导管的扫描电镜图；Fig. 1 is the scanning electron microscope picture of the braided catheter obtained in the step (1) of the embodiment of the present invention 1;
图2为本发明实施例1步骤(2)中得到的丝蛋白纤维-丝蛋白膜复合导管的扫描电镜图；2 is a scanning electron microscope image of the silk protein fiber-silk protein membrane composite catheter obtained in step (2) of Example 1 of the present invention;
图3为本发明实施例2中得到的丝蛋白神经导管的扫描电镜图；Fig. 3 is the scanning electron microscope picture of the silk protein nerve conduit obtained in the embodiment of the present invention 2;
图4为本发明实施例3中得到的丝蛋白神经导管内部取向丝蛋白海绵的扫描电镜图；Fig. 4 is the scanning electron microscope image of the oriented silk protein sponge inside the silk protein nerve conduit obtained in Example 3 of the present invention;
图5为本发明实施例3中得到的丝蛋白神经导管移植移植大鼠坐骨神经缺损后再生神经与自体移植的电生理波形图。Figure 5 is an electrophysiological waveform diagram of the regenerated nerve and autologous transplantation after the silk protein nerve conduit obtained in Example 3 of the present invention was transplanted into the rat sciatic nerve defect.
具体实施方式Detailed ways
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
本发明提供了一种丝蛋白神经导管，包括：导管部分与填充在导管部分内的填充海绵；所述导管部分包括导管基体与复合在导管基体表面的丝蛋白薄膜；所述导管基体由丝蛋白纤维编织形成；所述填充海绵由具有取向结构的丝蛋白纳米纤维凝胶形成。The invention provides a silk protein nerve conduit, comprising: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
本发明通过在丝蛋白纤维编织形成的导管基体表面复合丝蛋白薄膜，提高了导管的力学性能；导管内部设置有具有取向结构的丝蛋白纳米纤维海绵，具有与神经轴突方向一致的取向结构，能够提供取向诱导信号，促进神经细胞的迁移增值，也能指导神经纤维的再生和延伸，显著提高神经导管的功能；因此本发明通过丝蛋白管和丝蛋白取向海绵的结合，解决力学性能和取向诱导的问题。The invention improves the mechanical properties of the catheter by compounding the silk protein film on the surface of the catheter matrix formed by weaving the silk protein fibers; the silk protein nanofiber sponge with the orientation structure is arranged inside the catheter, and the orientation structure is consistent with the direction of the nerve axon. It can provide orientation-inducing signals, promote the migration and proliferation of nerve cells, and also guide the regeneration and extension of nerve fibers, significantly improving the function of nerve conduits; therefore, the present invention solves mechanical properties and orientation through the combination of silk protein tubes and silk protein orientation sponges. induced problems.
本发明还提供了一种上述丝蛋白神经导管的制备方法，包括：S1)将丝蛋白纤维进行编织得到导管基体；S2)将所述导管基体浸泡在丝蛋白溶液中，得到导管部分；S3)将具有取向结构的丝蛋白纳米纤维凝胶与导管部分结合，冷冻干燥后，得到丝蛋白神经导管。The present invention also provides a method for preparing the above silk protein nerve conduit, comprising: S1) weaving silk protein fibers to obtain a conduit matrix; S2) soaking the conduit matrix in a silk protein solution to obtain a conduit portion; S3) The silk protein nanofiber gel with oriented structure is combined with the conduit part, and after freeze-drying, the silk protein nerve conduit is obtained.
本发明对所有原料的来源并没有特殊的限制，为市售即可。The present invention does not have any special restrictions on the sources of all raw materials, which can be commercially available.
将丝蛋白纤维进行编织得到导管基体；所述丝蛋白纤维优选为蚕丝，更优选为脱胶蚕丝，再优选为80～100旦脱胶蚕丝；所述编织的编织角优选为45°～60°；所述编织的速度优选为30～60r/min，更优选为40～50r/min，再优选为45r/min；所述编织时的齿轮比优选为(60～100)：(20～60)，更优选为(70～90)：(30～50)，再优选为80：40；所述编织的骨架结构优选为针织结构或编 织结构；所述编织优选在光滑金属模具上进行；所述光滑金属模具的直径优选为0.5～15mm，使得得到的导管基体的内径优选为0.5～15mm。The fibroin fiber is braided to obtain a catheter matrix; the fibroin fiber is preferably silk, more preferably degummed silk, and more preferably degummed silk of 80-100 deniers; the braiding angle of the braiding is preferably 45°～60°; The speed of the weaving is preferably 30-60r/min, more preferably 40-50r/min, and more preferably 45r/min; the gear ratio during the weaving is preferably (60-100): (20-60), more preferably It is preferably (70-90):(30-50), more preferably 80:40; the woven skeleton structure is preferably a knitted structure or a woven structure; the weaving is preferably performed on a smooth metal mold; the smooth metal The diameter of the mold is preferably 0.5 to 15 mm, so that the inner diameter of the resulting catheter base is preferably 0.5 to 15 mm.
将所述导管基体浸泡在丝蛋白溶液中，优选将带有模具的导管基体浸泡在丝蛋白溶液中；所述丝蛋白溶液中丝蛋白的浓度优选为2～10wt％；在本发明提供的一些实施例中，所述丝蛋白溶液的浓度优选为2wt％；在本发明提供的一些实施例中，所述丝蛋白溶液的浓度优选为5wt％；在本发明提供的另一些实施例中，所述丝蛋白溶液的浓度优选为10wt％；所述丝蛋白溶液中的丝蛋白包括丝蛋白纳米纤维；所述丝蛋白纳米纤维的质量优选为丝蛋白总质量的0％～40％，更优选为丝蛋白总质量的0.5％～30％，再优选为丝蛋白总质量的0.5％～10％，最优选为丝蛋白总质量的0.8％～4％；利用丝蛋白纳米纤维诱导普通丝蛋白构象转变速率；所述丝蛋白纳米纤维的直径优选为10～20nm；所述丝蛋白纳米纤维的长度优选为1～2μm；所述导管基体与丝蛋白溶液的体积比优选为1：(1.5～3)，更优选为1：(2～2.5)，再优选为1：2；将导管基体浸泡在丝蛋白溶液中，优选在10℃～90℃蒸发，随着丝蛋白溶液中水分的缓慢蒸发，会在导管基体上形成一层不溶性丝蛋白膜，通过纳米纤维诱导和温度诱导的协同作用调控丝蛋白的构象转百年速率，最终使得薄膜稳定获得在水中的稳定性，制备出力学性能良好、在水中稳定存在的神经导管的导管部分。The catheter substrate is soaked in the silk protein solution, preferably the catheter substrate with the mold is soaked in the silk protein solution; the concentration of the silk protein in the silk protein solution is preferably 2-10wt%; In the embodiments, the concentration of the silk protein solution is preferably 2wt%; in some embodiments provided by the present invention, the concentration of the silk protein solution is preferably 5wt%; in other embodiments provided by the present invention, the The concentration of the silk protein solution is preferably 10wt%; the silk protein in the silk protein solution includes silk protein nanofibers; the quality of the silk protein nanofibers is preferably 0% to 40% of the total mass of the silk protein, more preferably 0.5% to 30% of the total mass of silk protein, preferably 0.5% to 10% of the total mass of silk protein, most preferably 0.8% to 4% of the total mass of silk protein; use silk protein nanofibers to induce the conformational transition of ordinary silk protein The diameter of the silk protein nanofibers is preferably 10-20 nm; the length of the silk protein nanofibers is preferably 1-2 μm; the volume ratio of the catheter matrix to the silk protein solution is preferably 1: (1.5-3) , more preferably 1:(2～2.5), more preferably 1:2; soak the catheter matrix in the silk protein solution, preferably evaporate at 10℃～90℃, with the slow evaporation of water in the silk protein solution, it will A layer of insoluble silk protein film is formed on the catheter substrate, and the conformational transition rate of silk protein is regulated by the synergistic effect of nanofiber induction and temperature induction, and finally the film is stabilized in water. The conduit portion of a stable nerve conduit.
将具有取向结构的丝蛋白纳米纤维凝胶与导管部分结合；在本发明中，可按照以下方法进行结合：将丝蛋白纳米纤维凝胶利用电场诱导，得到具有取向结构的丝蛋白纳米纤维凝胶；然后将导管部分沿着取向方向插进具有取向结构的丝蛋白纳米纤维凝胶中，使其充满导管部分；其中，所述丝蛋白纳米纤维凝胶优选按照以下步骤进行：将丝蛋白纳米纤维水溶液40℃～60℃浓缩至浓度为6～12wt％的第一丝蛋白纳米纤维水溶液；将第一丝蛋白纳米纤维水溶液在20℃～35℃浓缩至浓度为18～24wt％的第二丝蛋白纳米纤维水溶液；将所述第二丝蛋白纳米纤维水溶液加水稀释至浓度为0.2～4wt％的第三丝蛋白纳米纤维水溶液，然后在50℃～70℃密封孵育，得到丝蛋白纳米纤维凝胶；所述丝蛋白纳米纤维同上所述，在此不再赘述；所述电场诱导的电场强度优选为10～120V，更优选为10～100V；所述电场诱导的时间优选为5～60min；然后利用导管部分和凝胶力学性能的差异，将导管部分沿着凝胶取向方向***具有取向结 构的丝蛋白纳米纤维凝胶中，使得凝胶充满导管。The silk protein nanofiber gel with an orientation structure is combined with the catheter part; in the present invention, the combination can be performed according to the following method: the silk protein nanofiber gel is induced by an electric field to obtain a silk protein nanofiber gel with an orientation structure. Then insert the catheter part into the silk protein nanofiber gel with the orientation structure along the orientation direction, so that it fills the catheter part; wherein, the silk protein nanofiber gel is preferably carried out according to the following steps: the silk protein nanofiber The aqueous solution is concentrated at 40°C to 60°C to a first silk protein nanofiber aqueous solution with a concentration of 6-12wt%; the first silk protein nanofiber aqueous solution is concentrated at 20°C to 35°C to a concentration of 18-24wt% of the second silk protein Nanofiber aqueous solution; dilute the second silk protein nanofiber aqueous solution with water to a third silk protein nanofiber aqueous solution with a concentration of 0.2-4wt%, and then seal and incubate at 50°C to 70°C to obtain a silk protein nanofiber gel; The silk protein nanofibers are the same as described above, and will not be repeated here; the electric field strength induced by the electric field is preferably 10-120V, more preferably 10-100V; the electric field-induced time is preferably 5-60min; then use The difference in the mechanical properties of the catheter part and the gel, the catheter part was inserted into the silk protein nanofiber gel with an oriented structure along the gel orientation direction, so that the gel filled the catheter.
或者按以下步骤结合：将导管部分沿着平行于电极的方向浸入丝蛋白纳米纤维凝胶中，然后进行电场处理；所述丝蛋白纳米纤维凝胶同上所述，在此不再赘述；所述电场处理的强度优选为10～120V；所述电场处理的时间优选为5～60min；通过电场处理从而使得导管内部的纳米纤维定向移动，并在导管内部靠近正极的部分形成取向凝胶，所占体积大约为1/3，优选重复上述浸入与电场处理步骤，直到具有取向结构的丝蛋白纳米纤维凝胶充满导管部分90％以上。Or combined according to the following steps: the catheter part is immersed in the silk protein nanofiber gel along the direction parallel to the electrode, and then electric field treatment is performed; the silk protein nanofiber gel is as described above, and will not be repeated here; The intensity of the electric field treatment is preferably 10-120V; the time of the electric field treatment is preferably 5-60min; the electric field treatment makes the nanofibers inside the conduit move directionally, and forms an orientation gel in the portion near the positive electrode inside the conduit, which occupies The volume is about 1/3, and it is preferable to repeat the above steps of immersion and electric field treatment until the silk protein nanofiber gel with oriented structure fills more than 90% of the catheter part.
具有取向结构的丝蛋白纳米纤维凝胶与导管部分结合后，冷冻干燥，得到丝蛋白神经导管；所述冷冻干燥优选具体为：先在-20℃～-50℃冷冻1～12h后，然后在-2℃～-90℃冷冻干燥24～72h。通过冷冻干燥，使得凝胶转变为具有取向结构的多孔海绵，最终获得外部为导管，内部为取向海绵的复合神经导管。After the silk protein nanofiber gel with an oriented structure is combined with the catheter part, freeze-drying is performed to obtain a silk protein nerve catheter; the freeze-drying is preferably specifically: firstly freeze at -20°C to -50°C for 1 to 12 hours, and then freeze at -20°C to -50°C for 1 to 12 hours. -2℃～-90℃ freeze drying for 24～72h. Through freeze-drying, the gel is transformed into a porous sponge with an oriented structure, and finally a composite nerve conduit with a conduit on the outside and an oriented sponge on the inside is obtained.
本发明制备的丝蛋白神经导管没有加入任何有毒物质，其全部成分均为天然蛋白质，相容性好；并且通过调控丝蛋白的构象转变速率，在水溶液中直接稳定获得不溶于水的丝蛋白纤维-薄膜复合的导管部分，提高了导管的力学性能，使其满足临床应用需求，导管内部为取向丝蛋白纳米纤维海绵，具有与神经轴突方向一致的取向结构，能够提供取向诱导信号，促进神经细胞的迁移增值，也能指导神经纤维的再生和延伸，显著提高神经导管的功能；通过丝蛋白管和丝蛋白取向海绵的结合，解决力学性能和取向诱导的问题；再者，本发明整个技术均基于基础的物理处理过程，不存在化学反应和苛刻制备条件，有利于技术的产业转化和功能丰富，尽管是温和的物理调控，但其相关参数和工艺融合的关键步骤，均基于对丝蛋白本质的深入理解，特别是丝蛋白组装机制的深入理解，不同参数的整合共同决定导管最终性能，而非简单的物理方法的罗列，因此技术存在显著的创新性。The silk protein nerve conduit prepared by the invention does not add any toxic substances, all its components are natural proteins, and have good compatibility; and by regulating the conformational transition rate of the silk protein, the water-insoluble silk protein fiber can be directly and stably obtained in an aqueous solution. -The catheter part of the film composite improves the mechanical properties of the catheter and makes it meet the needs of clinical applications. The interior of the catheter is an oriented silk protein nanofiber sponge, which has an oriented structure consistent with the direction of the nerve axon, which can provide orientation-inducing signals and promote neural The migration and proliferation of cells can also guide the regeneration and extension of nerve fibers, and significantly improve the function of nerve conduits; through the combination of silk protein tubes and silk protein orientation sponges, the problems of mechanical properties and orientation induction are solved; All are based on the basic physical treatment process, there is no chemical reaction and harsh preparation conditions, which is conducive to the industrial transformation and functional enrichment of the technology. Although it is a mild physical control, its related parameters and key steps of process integration are all based on silk protein. The in-depth understanding of the essence, especially the in-depth understanding of the silk protein assembly mechanism, the integration of different parameters jointly determines the final performance of the catheter, rather than the list of simple physical methods, so the technology has significant innovation.
为了进一步说明本发明，以下结合实施例对本发明提供的一种丝蛋白神经导管及其制备方法进行详细描述。In order to further illustrate the present invention, the silk protein nerve conduit provided by the present invention and its preparation method are described in detail below with reference to the examples.
以下实施例中所用的试剂均为市售。The reagents used in the following examples are all commercially available.
实施例中所用丝蛋白纳米纤维的直径为10nm，长度为1μm；The silk protein nanofibers used in the examples have a diameter of 10 nm and a length of 1 μm;
实施例中丝蛋白纳米纤维凝胶的制备方法为：将丝蛋白纳米纤维(直径10～20nm，长度为1～2μm)为水溶液40℃～60℃浓缩至浓度为6～12wt％的第一丝蛋白纳米纤维水溶液；将第一丝蛋白纳米纤维水溶液在20℃～35℃浓缩至浓度为18～24wt％的第二丝蛋白纳米纤维水溶液；将所述第二丝蛋白纳米纤维水溶液加水稀释至浓度为0.2～4wt％的第三丝蛋白纳米纤维水溶液，然后在50℃～70℃密封孵育，得到丝蛋白纳米纤维凝胶；In the embodiment, the preparation method of the silk protein nanofiber gel is as follows: the silk protein nanofibers (10-20 nm in diameter, 1-2 μm in length) are concentrated into an aqueous solution at 40° C. to 60° C. to a first silk with a concentration of 6-12 wt %. Protein nanofiber aqueous solution; concentrate the first silk protein nanofiber aqueous solution at 20°C to 35°C to a second silk protein nanofiber aqueous solution with a concentration of 18-24 wt %; dilute the second silk protein nanofiber aqueous solution with water to the concentration A 0.2-4wt% aqueous solution of the third silk protein nanofibers, and then sealed and incubated at 50°C to 70°C to obtain a silk protein nanofiber gel;
所述实施例中的丝蛋白可根据Reversible Hydrogel-Solution System of Silk with High Beta-Sheet Content(Biomacromolecules 2014,15,3044-3051)制备。The silk protein in the described embodiment can be prepared according to Reversible Hydrogel-Solution System of Silk with High Beta-Sheet Content (Biomacromolecules 2014, 15, 3044-3051).
实施例1Example 1
(1)选取光滑的直径为3mm第一模具，用熟丝线100旦脱胶蚕丝，通过纺织机器在其上面织出骨架结构，编织角为45°，速度为45r/min，齿轮比为80：40；编织的骨架结构为编织结构。(1) Select a smooth first die with a diameter of 3mm, degumming silk with a 100-denier cooked silk thread, and weave a skeleton structure on it by a textile machine, the weaving angle is 45°, the speed is 45r/min, and the gear ratio is 80:40 ; The braided skeleton structure is a braided structure.
(2)将编织好的导管完全浸没于2％丝蛋白水溶液(丝蛋白纳米纤维的含量为丝蛋白总质量的0.8％)中，导管与丝蛋白水溶液的体积比为1：2，在10℃的条件下，随着丝蛋白溶液中水分的缓慢蒸发，会在骨架上形成一层不溶性的丝素蛋白膜，获得丝蛋白纤维-丝蛋白膜复合导管。(2) The braided catheter is completely immersed in a 2% silk protein aqueous solution (the content of silk protein nanofibers is 0.8% of the total mass of silk protein), and the volume ratio of the catheter to the silk protein aqueous solution is 1:2. Under certain conditions, with the slow evaporation of water in the silk protein solution, an insoluble silk fibroin film will be formed on the skeleton to obtain a silk protein fiber-silk protein film composite conduit.
(3)制备0.2％取向的丝蛋白纳米纤维凝胶，电场诱导的基本参数为：电场强度10V，处理时间5分钟。随后将丝蛋白纤维-丝蛋白膜复合导管沿着取向方向插进凝胶基质，使得凝胶充满导管内部。放入-20℃冰箱冷冻过夜，再放入冷冻干燥机中-80℃冷冻干燥48h取出，获得填充有取向丝蛋白海绵的丝蛋白神经导管。(3) Preparation of 0.2% oriented silk protein nanofiber gel, the basic parameters of electric field induction are: electric field intensity 10V, treatment time 5 minutes. The fibroin fiber-fibroin membrane composite catheter was then inserted into the gel matrix along the orientation direction, so that the gel filled the interior of the catheter. Put it into a -20°C refrigerator to freeze overnight, and then put it into a freeze dryer at -80°C for 48 hours to freeze-dry it and take out to obtain a silk protein nerve conduit filled with an oriented silk protein sponge.
利用扫描电子显微镜对步骤(1)中得到的编织的导管进行分析，得到其扫描电镜图，如图1所示。The braided catheter obtained in step (1) is analyzed with a scanning electron microscope, and its scanning electron microscope image is obtained, as shown in FIG. 1 .
利用扫描电子显微镜对步骤(2)中得到的丝蛋白纤维-丝蛋白膜复合导管进行分析，得到其扫描电镜图，如图2所示。The fibroin fiber-fibroin membrane composite conduit obtained in step (2) is analyzed by scanning electron microscope, and its scanning electron microscope image is obtained, as shown in FIG. 2 .
实施例2Example 2
(1)选取光滑的直径为5mm第一模具，用熟丝线100旦脱胶蚕丝，通过纺织机器在其上面织出骨架结构，编织角为60°，速度为45r/min，齿轮比为80：40；编织的骨架结构为编织结构。(1) Select a smooth first die with a diameter of 5mm, degummed silk with 100 denier of cooked silk thread, and weave a skeleton structure on it by a textile machine, the weaving angle is 60°, the speed is 45r/min, and the gear ratio is 80:40 ; The braided skeleton structure is a braided structure.
(2)将编织好的导管完全浸没于10％丝蛋白溶液(丝蛋白纳米纤维的含量为丝蛋白质量的4％)中，导管与丝蛋白水溶液的体积比为1：2，在90℃的条件下，随着丝素溶液中水分的缓慢蒸发，会在骨架上形成一层不溶性的丝素蛋白膜，获得丝蛋白纤维-丝蛋白膜复合导管。(2) The braided catheter is completely immersed in a 10% silk protein solution (the content of silk protein nanofibers is 4% of the silk protein mass), and the volume ratio of the catheter to the silk protein aqueous solution is 1:2. Under the conditions, along with the slow evaporation of water in the silk fibroin solution, an insoluble silk fibroin film will be formed on the skeleton to obtain a silk protein fiber-silk protein film composite conduit.
(3)将导管沿着平行于电极的方向放置并浸入4％丝蛋白纳米纤维凝胶中，随后对凝胶进行电场处理，电场强度100V，处理时间30min，从而使得导管内部的纳米纤维定向移动，并在导管内部靠近正极的部分形成取向凝胶，所占体积大约在1/3，随后继续在导管无取向凝胶的部分浸满凝胶，重复电场处理过程，直至90％以上的空间被取向凝胶占据，将导管取出，获得复合导管。放入-20℃冰箱冷冻过夜，再放入冷冻干燥机中-50℃冷冻干燥48h取出将凝胶转变为取向海绵，最终获得具有取向信号的丝蛋白神经导管。(3) The catheter was placed in a direction parallel to the electrodes and immersed in 4% silk protein nanofiber gel, and then the gel was subjected to electric field treatment, the electric field strength was 100V, and the treatment time was 30min, so as to make the nanofibers inside the catheter move directionally , and the oriented gel is formed in the part near the positive electrode inside the catheter, which occupies about 1/3 of the volume, and then continues to be filled with gel in the part of the catheter without oriented gel, and the electric field treatment process is repeated until more than 90% of the space is filled with gel. The orientation gel occupies, the catheter is removed, and the composite catheter is obtained. Put it in a -20°C refrigerator overnight, and then put it in a freeze dryer at -50°C for 48 hours to freeze-dry it out to convert the gel into an orientation sponge, and finally obtain a silk protein nerve conduit with an orientation signal.
利用扫描电子显微镜对实施例2中得到的丝蛋白神经导管进行分析，得到其扫描电镜图，如图3所示。The silk protein nerve conduit obtained in Example 2 was analyzed by scanning electron microscope, and its scanning electron microscope image was obtained, as shown in FIG. 3 .
实施例3Example 3
(1)选取光滑的直径为10mm第一模具，用熟丝线100旦脱胶蚕丝，通过纺织机器在其上面织出骨架结构，编织角为60°，速度为45r/min，齿轮比为80：40；编织的骨架结构为编织结构。(1) Select a smooth first die with a diameter of 10mm, degummed silk with a 100-denier cooked silk thread, and weave a skeleton structure on it by a textile machine. The weaving angle is 60°, the speed is 45r/min, and the gear ratio is 80:40 ; The braided skeleton structure is a braided structure.
(2)将编织好的导管完全浸没于5％丝蛋白溶液(丝蛋白纳米纤维的含量为丝蛋白总质量的2％)中，导管与丝蛋白水溶液的体积比为1：2，在90℃的条件下，随着丝素溶液中水分的缓慢蒸发，会在骨架上形成一层不溶性的丝素蛋白膜，获得丝蛋白纤维-丝蛋白膜复合导管。(2) The braided catheter is completely immersed in 5% silk protein solution (the content of silk protein nanofibers is 2% of the total mass of silk protein), and the volume ratio of the catheter to the silk protein aqueous solution is 1:2. Under certain conditions, with the slow evaporation of water in the silk fibroin solution, an insoluble silk fibroin film will be formed on the skeleton to obtain a silk protein fiber-silk protein film composite conduit.
(3)制备4％取向的丝蛋白纳米纤维凝胶，电场诱导的基本参数为：电场强度100V，处理时间60min。随后将复合丝蛋白导管沿着取向方向插进凝胶基质，使得凝胶充满导管内部。放入-20℃冰箱冷冻过夜，再放入冷冻干燥机中-80℃冷冻干燥48h取出，获得填充有取向丝蛋白海绵的丝蛋白神经导管。(3) Preparation of 4% oriented silk protein nanofiber gel, the basic parameters of electric field induction are: electric field intensity 100V, treatment time 60min. The composite fibroin conduit was then inserted into the gel matrix along the orientation direction so that the gel filled the interior of the conduit. Put it into a -20°C refrigerator to freeze overnight, and then put it into a freeze dryer at -80°C for 48 hours to freeze-dry it and take out to obtain a silk protein nerve conduit filled with an oriented silk protein sponge.
利用扫描电子显微镜对实施例3中得到的丝蛋白神经导管进行分析，得到其内部取向丝蛋白海绵的扫描电镜图，如图4所示。The silk protein nerve conduit obtained in Example 3 was analyzed with a scanning electron microscope, and a scanning electron microscope image of the internally oriented silk protein sponge was obtained, as shown in FIG. 4 .
将实施例3中得到的丝蛋白神经导管移植移植大鼠坐骨神经缺损后再生神经与自体移植的电生理波形图，如图5所示。神经导管移植到大鼠缺损部位 12周后检测到电信号传导。Figure 5 shows the electrophysiological waveforms of regenerated nerves and autologous transplantation after the silk protein nerve conduit obtained in Example 3 was transplanted into the rat sciatic nerve defect. Electrical signaling was detected 12 weeks after nerve conduit transplantation into the rat defect.
综上所述，本发明公开了一种取向丝素蛋白神经导管及其制备方法，从材料和仿生结构设计入手，设计了分别由蚕丝基导管和取向丝蛋白纳米纤维支架复合的神经导管。将蚕丝编织纤维增强的丝蛋白导管利用具有取向结构的丝蛋白凝胶进行填充，通过冷冻干燥技术将凝胶转变成取向海绵，获得具有取向诱导信号的神经导管。本发明所述制备过程条件温和，创新性地将不同丝蛋白制备技术有机结合，获得具有取向信号，力学性能满足应用需求的神经导管，修复性能同人自体神经相当，具有良好的临床应用价值。In summary, the present invention discloses an oriented silk fibroin nerve conduit and a preparation method thereof. Starting from the design of materials and biomimetic structures, a nerve conduit which is respectively composed of a silk-based conduit and an oriented silk protein nanofiber scaffold is designed. The silk protein conduit reinforced by silk woven fibers is filled with silk protein gel with an orientation structure, and the gel is transformed into an orientation sponge by freeze-drying technology to obtain a nerve conduit with orientation-inducing signals. The preparation process of the invention has mild conditions, and innovatively combines different silk protein preparation technologies to obtain a nerve conduit with orientation signals and mechanical properties that meet application requirements.

Claims (10)

  1. 一种丝蛋白神经导管，其特征在于，包括：导管部分与填充在导管部分内的填充海绵；所述导管部分包括导管基体与复合在导管基体表面的丝蛋白薄膜；所述导管基体由丝蛋白纤维编织形成；所述填充海绵由具有取向结构的丝蛋白纳米纤维凝胶形成。A silk protein nerve conduit, characterized in that it comprises: a conduit part and a filling sponge filled in the conduit part; the conduit part comprises a conduit base and a silk protein film compounded on the surface of the conduit base; the conduit base is made of silk protein The fibers are woven and formed; the filling sponge is formed of a silk protein nanofiber gel with an oriented structure.
  2. 一种丝蛋白神经导管的制备方法，其特征在于，包括：A preparation method of silk protein nerve conduit, is characterized in that, comprises:
    S1)将丝蛋白纤维进行编织得到导管基体；S1) weaving silk protein fibers to obtain a catheter matrix;
    S2)将所述导管基体浸泡在丝蛋白溶液中，得到导管部分；S2) soaking the catheter matrix in the silk protein solution to obtain the catheter part;
    S3)将具有取向结构的丝蛋白纳米纤维凝胶与导管部分结合，冷冻干燥后，得到丝蛋白神经导管。S3) combining the silk protein nanofiber gel with an oriented structure with the catheter part, and after freeze-drying, the silk protein nerve catheter is obtained.
  3. 根据权利要求2所述的制备方法，其特征在于，所述丝蛋白纤维为80～100旦脱胶蚕丝；所述编织的编织角为45°～60°；编织速度为30～60r/min；所述导管基体的内径为0.5～15mm。The preparation method according to claim 2, wherein the fibroin fiber is 80-100 denier degummed silk; the weaving angle of the weaving is 45°-60°; the weaving speed is 30-60 r/min; The inner diameter of the catheter base body is 0.5-15 mm.
  4. 根据权利要求2所述的制备方法，其特征在于，所述丝蛋白溶液中丝蛋白的浓度为2～10wt％；所述丝蛋白包括丝蛋白纳米纤维；所述丝蛋白纳米纤维的质量为丝蛋白质量的0％～40％。The preparation method according to claim 2, wherein the concentration of silk protein in the silk protein solution is 2-10 wt%; the silk protein comprises silk protein nanofibers; the quality of the silk protein nanofibers is silk 0% to 40% of the protein content.
  5. 根据权利要求3所述的制备方法，其特征在于，所述丝蛋白纳米纤维的直径为10～20nm；长度为1～2μm。The preparation method according to claim 3, wherein the silk protein nanofibers have a diameter of 10-20 nm and a length of 1-2 μm.
  6. 根据权利要求2所述的制备方法，其特征在于，所述S2)具体为：preparation method according to claim 2, is characterized in that, described S2) is specially:
    将所述导管基体浸泡在丝蛋白溶液中，在10℃～90℃蒸发，得到导管部分。The catheter substrate is soaked in the silk protein solution and evaporated at 10°C to 90°C to obtain the catheter part.
  7. 根据权利要求2所述的制备方法，其特征在于，所述丝蛋白纳米纤维凝胶按照以下步骤进行制备：The preparation method according to claim 2, wherein the silk protein nanofiber gel is prepared according to the following steps:
    将丝蛋白纳米纤维水溶液40℃～60℃浓缩至浓度为6～12wt％的第一丝蛋白纳米纤维水溶液；Concentrating the silk protein nanofiber aqueous solution at 40°C to 60°C to a first silk protein nanofiber aqueous solution with a concentration of 6 to 12wt%;
    将第一丝蛋白纳米纤维水溶液在20℃～35℃浓缩至浓度为18～24wt％的第二丝蛋白纳米纤维水溶液；Concentrating the first silk protein nanofiber aqueous solution at 20°C to 35°C to a second silk protein nanofiber aqueous solution with a concentration of 18 to 24 wt %;
    将所述第二丝蛋白纳米纤维水溶液加水稀释至浓度为0.2～4wt％的第三丝 蛋白纳米纤维水溶液，然后在50℃～70℃密封孵育，得到丝蛋白纳米纤维凝胶。The second silk protein nanofiber aqueous solution is diluted with water to a third silk protein nanofiber aqueous solution with a concentration of 0.2-4wt%, and then sealed and incubated at 50°C to 70°C to obtain a silk protein nanofiber gel.
  8. 根据权利要求2所述的制备方法，其特征在于，所述步骤S3)具体为：The preparation method according to claim 2, wherein the step S3) is specifically:
    将丝蛋白纳米纤维凝胶利用电场诱导，得到具有取向结构的丝蛋白纳米纤维凝胶；The silk protein nanofiber gel is induced by an electric field to obtain a silk protein nanofiber gel with an oriented structure;
    然后将导管部分沿着取向方向插进具有取向结构的丝蛋白纳米纤维凝胶中，使其充满导管部分，取出后，冷冻干燥，得到丝蛋白神经导管；Then, the catheter part is inserted into the silk protein nanofiber gel with an oriented structure along the orientation direction, so that the catheter part is filled, and after being taken out, freeze-drying is performed to obtain the silk protein nerve catheter;
    或者：将导管部分沿着平行于电极的方向浸入丝蛋白纳米纤维凝胶中，然后进行电场处理，取出后，冷冻干燥，得到丝蛋白神经导管。Or: the catheter part is immersed in the silk protein nanofiber gel along the direction parallel to the electrode, then subjected to electric field treatment, taken out, and freeze-dried to obtain the silk protein nerve catheter.
  9. 根据权利要求8所述的制备方法，其特征在于，所述电场诱导与电场处理的电场强度各自独立地为10～120V；时间各自独立地为5～60min。The preparation method according to claim 8, wherein the electric field strengths of the electric field induction and the electric field treatment are independently 10-120V, and the time is independently 5-60min.
  10. 根据权利要求2所述的制备方法，其特征在于，所述冷冻干燥具体为：先在-20℃～-50℃冷冻1～12h后，然后在-2℃～-90℃冷冻干燥24～72h。The preparation method according to claim 2, wherein the freeze-drying is specifically: firstly freeze-drying at -20°C～-50°C for 1～12h, and then freeze-drying at -2°C～-90°C for 24～72h .
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