CN112263714A - Silk protein nerve conduit and preparation method thereof - Google Patents
Silk protein nerve conduit and preparation method thereof Download PDFInfo
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- CN112263714A CN112263714A CN202011102746.5A CN202011102746A CN112263714A CN 112263714 A CN112263714 A CN 112263714A CN 202011102746 A CN202011102746 A CN 202011102746A CN 112263714 A CN112263714 A CN 112263714A
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- 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
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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
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
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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
- A61L27/50—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
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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
- A61L27/50—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
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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
- A61L27/50—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
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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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
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/02—Methods for coating medical devices
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
The invention provides a silk protein nerve conduit, which comprises: the conduit part and the filling sponge filled in the conduit part; the catheter part comprises a catheter matrix and a silk fibroin film compounded on the surface of the catheter matrix; the catheter matrix is formed by weaving silk protein fibers; the filled sponge is formed by silk protein nanofiber gel with an oriented structure. Compared with the prior art, the silk protein nanofiber sponge with the oriented structure is arranged in the catheter, has the oriented structure consistent with the direction of the nerve axon, can provide an oriented induction signal, promotes the migration and proliferation of nerve cells, can also guide the regeneration and extension of nerve fibers, and obviously improves the function of the nerve catheter; therefore, the invention solves the problems of mechanical property and orientation induction by combining the silk protein tube and the silk protein oriented sponge.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a fibroin nerve conduit and a preparation method thereof.
Background
Peripheral nerve injury is a clinically common condition, autografting is often used to bridge peripheral nerve defects, however limited availability and donor site morbidity still exists, and researchers have assisted peripheral nerve regeneration by developing biomaterial-based nerve conduits to overcome this limitation. Artificial catheters have proven to be capable of filling the nerve spaces, synthetic nerve catheters are expected to replace autografts, and improvements in biomaterials have great potential in promoting nerve regeneration and functional recovery. Therefore, the developed nerve conduit not only has good biological performance, but also should provide a better microenvironment for the regeneration of nerve tissues.
Nerve conduits made of various materials have been developed, and have relatively good effects in animal experiments and clinical researches, but the performance of the nerve conduits is still greatly different from that of autologous nerve conduits. How to introduce different inducing signals into the design of the nerve conduit according to the bionic concept is an effective way for further optimizing the function of the nerve conduit and improving the peripheral nerve injury repair.
Due to excellent biocompatibility, degradability and low inflammatory reaction, silk protein is widely applied to repair and regeneration of different tissues, and good early results are obtained in the field of nerve repair. At present, researchers mainly realize the introduction of orientation signals through an electrospinning technology and a directional freezing technology, the effective combination of the orientation signals and a nerve conduit and the stability of the technology have great problems, and a new preparation method needs to be considered to obtain a silk protein conduit with better orientation induction signals.
In the research of our team, a composite catheter with good mechanical property is successfully obtained by combining a silk protein weaving technology and a silk protein film forming technology, and is applied to the substitution of small blood vessels, however, due to the instability of a silk protein solution, the technical improvement and breakthrough are still needed on the basis of understanding the silk protein mechanism if a water-insoluble silk protein fiber-film composite catheter is stably obtained; in addition, silk protein gel or sponge with an oriented structure is successfully obtained by inducing directional migration of silk protein nanofibers through an electric field, and is successfully applied to wound repair and induced migration of nerve cells, but the gel or sponge has poor mechanical properties and is difficult to meet the requirements of clinical application, so that the composite nerve conduit with excellent mechanical properties at the outer part and application requirements met and the oriented silk protein sponge at the inner part and capable of providing orientation induction signals is prepared based on deep understanding of silk protein by teams, and is expected to be prepared through combination and optimization of different technologies so as to achieve better repair effect. The key point of the invention is to solve the problem of insoluble and stable acquisition of the silk fibroin braided tube and the silk fibroin film in the water phase environment and the technical problem of efficiently introducing the oriented sponge into the catheter.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a silk fibroin nerve conduit with orientation-inducing signal and a preparation method thereof.
The invention provides a silk protein nerve conduit, which comprises: the conduit part and the filling sponge filled in the conduit part; the catheter part comprises a catheter matrix and a silk fibroin film compounded on the surface of the catheter matrix; the catheter matrix is formed by weaving silk protein fibers; the filled sponge is formed by silk protein nanofiber gel with an oriented structure.
The invention also provides a preparation method of the silk fibroin nerve conduit, which comprises the following steps:
s1) weaving silk protein fibers to obtain a catheter matrix;
s2) soaking the catheter substrate in silk protein solution to obtain a catheter part;
s3) combining the silk protein nanofiber gel with the oriented structure with the catheter part, and freeze-drying to obtain the silk protein nerve catheter.
Preferably, the silk protein fiber is 80-100 denier degummed silk; the weaving angle of the weaving is 45-60 degrees; the weaving speed is 30-60 r/min; the inner diameter of the catheter base body is 0.5-15 mm.
Preferably, the concentration of the silk protein in the silk protein solution is 2-10 wt%; the silk protein comprises silk protein nanofibers; the mass of the silk fibroin nanofiber is 0% -40% of the mass of silk fibroin.
Preferably, the diameter of the silk protein nanofiber is 10-20 nm; the length is 1 to 2 μm.
Preferably, the S2) is specifically:
and soaking the catheter matrix in silk protein solution, and evaporating at 10-90 ℃ to obtain the catheter part.
Preferably, the silk protein nanofiber gel is prepared according to the following steps:
concentrating the silk fibroin nanofiber aqueous solution at 40-60 ℃ to obtain a first silk fibroin nanofiber aqueous solution with the concentration of 6-12 wt%;
concentrating the first silk protein nanofiber aqueous solution at 20-35 ℃ to obtain a second silk protein nanofiber aqueous solution with the concentration of 18-24 wt%;
and adding water to dilute the second silk protein nanofiber aqueous solution to a third silk protein nanofiber aqueous solution with the concentration of 0.2-4 wt%, and then carrying out sealed incubation at 50-70 ℃ to obtain the silk protein nanofiber gel.
Preferably, the step S3) is specifically:
inducing the silk fibroin nanofiber gel by using an electric field to obtain silk fibroin nanofiber gel with an oriented structure;
then inserting the conduit part into the silk fibroin nanofiber gel with an oriented structure along the orientation direction to fill the conduit part with the silk fibroin nanofiber gel, taking out the silk fibroin nanofiber gel, and freeze-drying to obtain a silk fibroin nerve conduit;
or: and immersing the catheter part into the silk protein nanofiber gel along the direction parallel to the electrodes, then carrying out electric field treatment, taking out, and carrying out freeze drying to obtain the silk protein nerve catheter.
Preferably, the electric field intensity of the electric field induction and the electric field treatment is 10-120V respectively and independently; the time is 5-60 min independently.
Preferably, the freeze-drying is specifically: freezing at-20 to-50 ℃ for 1 to 12 hours, and then freeze-drying at-2 to-90 ℃ for 24 to 72 hours.
The invention provides a silk protein nerve conduit, which comprises: the conduit part and the filling sponge filled in the conduit part; the catheter part comprises a catheter matrix and a silk fibroin film compounded on the surface of the catheter matrix; the catheter matrix is formed by weaving silk protein fibers; the filled sponge is formed by silk protein nanofiber gel with an oriented structure. Compared with the prior art, the silk protein nanofiber sponge with the oriented structure is arranged in the catheter, has the oriented structure consistent with the direction of the nerve axon, can provide an oriented induction signal, promotes the migration and proliferation of nerve cells, can also guide the regeneration and extension of nerve fibers, and obviously improves the function of the nerve catheter; therefore, the invention solves the problems of mechanical property and orientation induction by combining the silk protein tube and the silk protein oriented sponge.
Drawings
FIG. 1 is a scanning electron microscope image of a braided catheter obtained in step (1) of example 1 of the present invention;
FIG. 2 is a scanning electron microscope image of the silk fibroin fiber-silk fibroin film composite conduit obtained in step (2) of example 1 of the present invention;
FIG. 3 is a scanning electron micrograph of a fibroin nerve conduit obtained in example 2 of the present invention;
FIG. 4 is a scanning electron microscope image of oriented silk protein sponge inside the silk protein nerve conduit obtained in example 3 of the present invention;
FIG. 5 is an electrophysiology waveform diagram of regenerated nerve and autograft after sciatic nerve defect of transplanted rat with silk protein nerve conduit graft obtained in example 3 of the present invention.
Detailed Description
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, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a silk protein nerve conduit, which comprises: the conduit part and the filling sponge filled in the conduit part; the catheter part comprises a catheter matrix and a silk fibroin film compounded on the surface of the catheter matrix; the catheter matrix is formed by weaving silk protein fibers; the filled sponge is formed by silk protein nanofiber gel with an oriented structure.
The mechanical property of the catheter is improved by compounding the silk fibroin film on the surface of the catheter matrix formed by weaving silk fibroin fibers; the inside of the catheter is provided with the silk protein nanofiber sponge with an oriented structure, the silk protein nanofiber sponge with an oriented structure is provided with an oriented structure consistent with the direction of a nerve axon, an orientation induction signal can be provided, the migration and proliferation of nerve cells are promoted, the regeneration and extension of nerve fibers can be guided, and the function of the nerve catheter is obviously improved; therefore, the invention solves the problems of mechanical property and orientation induction by combining the silk protein tube and the silk protein oriented sponge.
The invention also provides a preparation method of the silk protein nerve conduit, which comprises the following steps: s1) weaving silk protein fibers to obtain a catheter matrix; s2) soaking the catheter substrate in silk protein solution to obtain a catheter part; s3) combining the silk protein nanofiber gel with the oriented structure with the catheter part, and freeze-drying to obtain the silk protein nerve catheter.
The present invention is not particularly limited in terms of the source of all raw materials, and may be commercially available.
Weaving silk protein fibers to obtain a catheter matrix; the silk protein fiber is preferably silk, more preferably degummed silk, and further preferably 80-100 denier degummed silk; the knitting angle of the knitting is preferably 45-60 degrees; the weaving speed is preferably 30-60 r/min, more preferably 40-50 r/min, and further preferably 45 r/min; the gear ratio during weaving is preferably (60-100): (20-60), more preferably (70-90): (30-50), and more preferably 80: 40; the braided framework structure is preferably a knitted structure or a braided structure; the weaving is preferably carried out on a smooth metal mold; the diameter of the smooth metal die is preferably 0.5-15 mm, so that the inner diameter of the obtained conduit base body is preferably 0.5-15 mm.
Soaking the catheter matrix in a silk fibroin solution, preferably soaking the catheter matrix with the mold in the silk fibroin solution; the concentration of the silk protein in the silk protein solution is preferably 2-10 wt%; in some embodiments provided herein, the concentration of the silk protein solution is preferably 2 wt%; in some embodiments provided herein, the concentration of the silk protein solution is preferably 5 wt%; in other embodiments provided herein, the concentration of the silk protein solution is preferably 10 wt%; the silk protein in the silk protein solution comprises silk protein nanofibers; the mass of the silk fibroin nanofiber is preferably 0-40% of the total mass of silk fibroin, more preferably 0.5-30% of the total mass of silk fibroin, still more preferably 0.5-10% of the total mass of silk fibroin, and most preferably 0.8-4% of the total mass of silk fibroin; inducing a common silk protein conformation transformation rate by utilizing the silk protein nanofiber; the diameter of the silk fibroin nanofiber is preferably 10-20 nm; the length of the silk protein nanofiber 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), preferably 1: 2; soaking the catheter matrix in a silk protein solution, preferably evaporating at 10-90 ℃, forming an insoluble silk protein film on the catheter matrix along with slow evaporation of water in the silk protein solution, regulating and controlling the conformational transition rate of silk protein through the synergistic effect of nanofiber induction and temperature induction, and finally enabling the film to stably obtain the stability in water to prepare the catheter part of the nerve catheter with good mechanical property and stable existence in water.
Combining the silk protein nanofiber gel having the oriented structure with the catheter portion; in the present invention, the combination can be performed as follows: inducing the silk fibroin nanofiber gel by using an electric field to obtain silk fibroin nanofiber gel with an oriented structure; then inserting the conduit part into the silk protein nanofiber gel with an oriented structure along the orientation direction to fill the conduit part; wherein, the silk protein nanofiber gel is preferably prepared according to the following steps: concentrating the silk fibroin nanofiber aqueous solution at 40-60 ℃ to obtain a first silk fibroin nanofiber aqueous solution with the concentration of 6-12 wt%; concentrating the first silk protein nanofiber aqueous solution at 20-35 ℃ to obtain a second silk protein nanofiber aqueous solution with the concentration of 18-24 wt%; adding water to dilute the second silk protein nanofiber aqueous solution to a third silk protein nanofiber aqueous solution with the concentration of 0.2-4 wt%, and then carrying out sealed incubation at 50-70 ℃ to obtain silk protein nanofiber gel; the silk protein nanofiber is the same as the silk protein nanofiber, and the description is omitted; the electric field intensity induced by the electric field is preferably 10-120V, and more preferably 10-100V; the time for inducing the electric field is preferably 5-60 min; and then the catheter part is inserted into the silk protein nanofiber gel with an oriented structure along the gel orientation direction by utilizing the difference of the mechanical properties of the catheter part and the gel, so that the gel is filled in the catheter.
Or the following steps are combined: immersing the catheter part into silk protein nanofiber gel along the direction parallel to the electrodes, and then carrying out electric field treatment; the silk protein nanofiber gel is the same as the silk protein nanofiber gel, and is not described in detail herein; the strength of the electric field treatment is preferably 10-120V; the time for the electric field treatment is preferably 5-60 min; and (3) performing electric field treatment to make the nano fibers in the catheter move directionally, and forming an oriented gel in the part, close to the positive electrode, in the catheter, wherein the occupied volume is about 1/3, and preferably repeating the steps of immersing and the electric field treatment until the silk protein nano fiber gel with an oriented structure is filled in more than 90% of the catheter part.
Combining the silk fibroin nanofiber gel with the orientation structure with the catheter part, and then freezing and drying to obtain a silk fibroin nerve catheter; the freeze-drying is preferably as follows: freezing at-20 to-50 ℃ for 1 to 12 hours, and then freeze-drying at-2 to-90 ℃ for 24 to 72 hours. And (3) freeze-drying to convert the gel into a porous sponge with an oriented structure, and finally obtaining the composite nerve conduit with the conduit at the outer part and the oriented sponge at the inner part.
The silk protein nerve conduit prepared by the invention is not added with any toxic substance, and all components are natural protein, so that the compatibility is good; the water-insoluble silk protein fiber-thin film composite catheter part is directly and stably obtained in an aqueous solution by regulating the conformational transition rate of silk protein, the mechanical property of the catheter is improved, and the catheter meets the requirements of clinical application; the problems of mechanical property and orientation induction are solved by combining the silk protein tube and the silk protein oriented sponge; moreover, the whole technology of the invention is based on the basic physical treatment process, chemical reaction and harsh preparation conditions do not exist, the industrial transformation and rich functions of the technology are facilitated, although mild physical regulation and control are realized, the key steps of relevant parameters and process fusion are based on deep understanding of silk protein essence, particularly silk protein assembly mechanism, and integration of different parameters jointly determines the final performance of the catheter, but not the list of simple physical methods, so the technology has obvious innovation.
In order to further illustrate the present invention, a silk protein nerve conduit and a method for preparing the same according to the present invention will be described in detail with reference to the following examples.
The reagents used in the following examples are all commercially available.
The diameter of the silk protein nanofiber used in the embodiment is 10nm, and the length is 1 μm;
the preparation method of the silk fibroin nanofiber gel in the embodiment comprises the following steps: concentrating silk fibroin nanofiber (with the diameter of 10-20 nm and the length of 1-2 mu m) into a first silk fibroin nanofiber aqueous solution with the concentration of 6-12 wt% at 40-60 ℃; concentrating the first silk protein nanofiber aqueous solution at 20-35 ℃ to obtain a second silk protein nanofiber aqueous solution with the concentration of 18-24 wt%; adding water to dilute the second silk protein nanofiber aqueous solution to a third silk protein nanofiber aqueous solution with the concentration of 0.2-4 wt%, and then carrying out sealed incubation at 50-70 ℃ to obtain silk protein nanofiber gel;
the silk protein in the examples can be prepared according to Reversible Hydrogel-Solution System of Silk with High Beta-Sheet Content (Biomacromolecules 2014,15, 3044-.
Example 1
(1) Selecting a smooth first die with the diameter of 3mm, degumming silk with 100 denier of boiled silk threads, weaving a skeleton structure on the silk by a textile machine, wherein the weaving angle is 45 degrees, the speed is 45r/min, and the gear ratio is 80: 40; the woven skeleton structure is a woven structure.
(2) Completely immersing the braided catheter in a 2% silk protein aqueous solution (the content of silk protein nano fibers is 0.8% of the total mass of silk protein), wherein the volume ratio of the catheter to the silk protein aqueous solution is 1: 2, under the condition of 10 ℃, a layer of insoluble silk fibroin film is formed on the framework along with the slow evaporation of the water in the silk fibroin solution, and the silk fibroin fiber-silk fibroin film composite conduit is obtained.
(3) Preparing 0.2% oriented silk protein nanofiber gel, and the basic parameters of electric field induction are as follows: the electric field strength was 10V and the treatment time was 5 minutes. The silk fibroin fiber-silk fibroin membrane composite catheter is then inserted into the gel matrix along the orientation direction such that the gel fills the interior of the catheter. Freezing in a refrigerator at-20 deg.C overnight, freeze drying in a freeze drier at-80 deg.C for 48 hr, and taking out to obtain the silk protein nerve conduit filled with oriented silk protein sponge.
Analyzing the braided catheter obtained in step (1) by using a scanning electron microscope to obtain a scanning electron microscope image, which is shown in fig. 1.
And (3) analyzing the silk fibroin fiber-silk fibroin film composite catheter obtained in the step (2) by using a scanning electron microscope to obtain a scanning electron microscope image, which is shown in fig. 2.
Example 2
(1) Selecting a smooth first die with the diameter of 5mm, degumming silk by using a boiled silk thread with the size of 100 denier, weaving a skeleton structure on the silk by a textile machine, wherein the weaving angle is 60 degrees, the speed is 45r/min, and the gear ratio is 80: 40; the woven skeleton structure is a woven structure.
(2) Completely immersing the braided catheter in a 10% silk protein solution (the content of silk protein nanofibers is 4% of the mass of silk protein), wherein the volume ratio of the catheter to the silk protein solution is 1: 2, under the condition of 90 ℃, a layer of insoluble silk fibroin film is formed on the framework along with the slow evaporation of water in the silk fibroin solution, and the silk fibroin fiber-silk fibroin film composite conduit is obtained.
(3) Placing the catheter in a direction parallel to the electrodes and immersing the catheter in 4% silk protein nanofiber gel, then carrying out electric field treatment on the gel, wherein the electric field intensity is 100V, the treatment time is 30min, so that the nanofibers inside the catheter move directionally, an oriented gel is formed in the part, close to the positive electrode, inside the catheter, the occupied volume is about 1/3, then continuing to immerse the part, without the oriented gel, of the catheter in the gel, repeating the electric field treatment process until more than 90% of the space is occupied by the oriented gel, and taking out the catheter to obtain the composite catheter. Freezing in a refrigerator at-20 deg.C overnight, freeze drying in a freeze drier at-50 deg.C for 48 hr, and converting the gel into oriented sponge to obtain silk protein nerve conduit with oriented signal.
The fibroin nerve conduits obtained in example 2 were analyzed by scanning electron microscopy to obtain a scanning electron micrograph, which is shown in fig. 3.
Example 3
(1) Selecting a first smooth die with the diameter of 10mm, degumming silk by using a boiled silk thread with the size of 100 denier, weaving a skeleton structure on the first die by a textile machine, wherein the weaving angle is 60 degrees, the speed is 45r/min, and the gear ratio is 80: 40; the woven skeleton structure is a woven structure.
(2) Completely immersing the braided catheter in a 5% silk protein solution (the content of silk protein nanofibers is 2% of the total mass of silk protein), wherein the volume ratio of the catheter to the silk protein solution is 1: 2, under the condition of 90 ℃, a layer of insoluble silk fibroin film is formed on the framework along with the slow evaporation of water in the silk fibroin solution, and the silk fibroin fiber-silk fibroin film composite conduit is obtained.
(3) Preparing 4% oriented silk protein nanofiber gel, and the basic parameters of electric field induction are as follows: the electric field intensity is 100V, and the processing time is 60 min. The composite silk fibroin catheter is then inserted into the gel matrix in an orientation such that the gel fills the interior of the catheter. Freezing in a refrigerator at-20 deg.C overnight, freeze drying in a freeze drier at-80 deg.C for 48 hr, and taking out to obtain the silk protein nerve conduit filled with oriented silk protein sponge.
The fibroin nerve conduits obtained in example 3 were analyzed by a scanning electron microscope to obtain a scanning electron microscope image of the internally oriented fibroin sponge, as shown in fig. 4.
Electrophysiology waveforms of regenerated nerves and autografts of transplanted rat sciatic nerve after transplantation of silk protein nerve conduit obtained in example 3 to sciatic nerve defect are shown in fig. 5. Electrical signal transmission was detected 12 weeks after the nerve conduit was transplanted to the rat defect site.
In summary, the invention discloses an oriented silk fibroin nerve conduit and a preparation method thereof, and the nerve conduit compounded by a silk-based conduit and an oriented silk fibroin nanofiber scaffold is designed by starting from material and bionic structure design. Filling the silk woven fiber reinforced silk protein catheter with silk protein gel with an oriented structure, and converting the gel into an oriented sponge by a freeze drying technology to obtain the nerve catheter with an orientation induction signal. The preparation process has mild conditions, creatively and organically combines different fibroin preparation technologies, obtains the nerve conduit with an orientation signal and the mechanical property meeting the application requirement, has the repairing property equivalent to that of the autologous nerve of a human body, and has good clinical application value.
Claims (10)
1. A silk protein nerve conduit, comprising: the conduit part and the filling sponge filled in the conduit part; the catheter part comprises a catheter matrix and a silk fibroin film compounded on the surface of the catheter matrix; the catheter matrix is formed by weaving silk protein fibers; the filled sponge is formed by silk protein nanofiber gel with an oriented structure.
2. A method for preparing a silk fibroin nerve conduit is characterized by comprising the following steps:
s1) weaving silk protein fibers to obtain a catheter matrix;
s2) soaking the catheter substrate in silk protein solution to obtain a catheter part;
s3) combining the silk protein nanofiber gel with the oriented structure with the catheter part, and freeze-drying to obtain the silk protein nerve catheter.
3. The preparation method according to claim 2, wherein the silk protein fiber is 80-100 denier degummed silk; the weaving angle of the weaving is 45-60 degrees; the weaving speed is 30-60 r/min; the inner diameter of the catheter base body is 0.5-15 mm.
4. 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 mass of the silk fibroin nanofiber is 0% -40% of the mass of silk fibroin.
5. The preparation method according to claim 3, wherein the silk protein nanofiber has a diameter of 10 to 20 nm; the length is 1 to 2 μm.
6. The preparation method according to claim 2, wherein S2) is specifically:
and soaking the catheter matrix in silk protein solution, and evaporating at 10-90 ℃ to obtain the catheter part.
7. The method of claim 2, wherein the silk fibroin nanofiber gel is prepared by the steps of:
concentrating the silk fibroin nanofiber aqueous solution at 40-60 ℃ to obtain a first silk fibroin nanofiber aqueous solution with the concentration of 6-12 wt%;
concentrating the first silk protein nanofiber aqueous solution at 20-35 ℃ to obtain a second silk protein nanofiber aqueous solution with the concentration of 18-24 wt%;
and adding water to dilute the second silk protein nanofiber aqueous solution to a third silk protein nanofiber aqueous solution with the concentration of 0.2-4 wt%, and then carrying out sealed incubation at 50-70 ℃ to obtain the silk protein nanofiber gel.
8. The preparation method according to claim 2, wherein the step S3) is specifically:
inducing the silk fibroin nanofiber gel by using an electric field to obtain silk fibroin nanofiber gel with an oriented structure;
then inserting the conduit part into the silk fibroin nanofiber gel with an oriented structure along the orientation direction to fill the conduit part with the silk fibroin nanofiber gel, taking out the silk fibroin nanofiber gel, and freeze-drying to obtain a silk fibroin nerve conduit;
or: and immersing the catheter part into the silk protein nanofiber gel along the direction parallel to the electrodes, then carrying out electric field treatment, taking out, and carrying out freeze drying to obtain the silk protein nerve catheter.
9. The method according to claim 8, wherein the electric field induction and the electric field treatment are each independently 10 to 120V in electric field strength; the time is 5-60 min independently.
10. The method according to claim 2, wherein the freeze-drying is in particular: freezing at-20 to-50 ℃ for 1 to 12 hours, and then freeze-drying at-2 to-90 ℃ for 24 to 72 hours.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113180885A (en) * | 2021-04-01 | 2021-07-30 | 重庆市畜牧科学院 | Optic nerve interface and its preparing method |
| WO2022077878A1 (en) * | 2020-10-15 | 2022-04-21 | 苏州大学 | Silk protein nerve conduit and preparation method therefor |
| CN117018290A (en) * | 2023-10-10 | 2023-11-10 | 常州丝波敦生物科技有限公司 | Silk protein nanofiber nerve conduit with bionic orientation structure and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010036992A2 (en) * | 2008-09-26 | 2010-04-01 | Trustees Of Tufts College | Active silk muco-adhesives, silk electrogelation process, and devices |
| CN107952112A (en) * | 2017-12-07 | 2018-04-24 | 武汉纺织大学 | A kind of nerve trachea of the function of directional guide containing silk fibroin nano-fiber and preparation method thereof |
| CN108114315A (en) * | 2018-02-22 | 2018-06-05 | 南通大学 | A kind of mariages fibroin nerve graft |
| CN110251732A (en) * | 2019-06-18 | 2019-09-20 | 南通纺织丝绸产业技术研究院 | A kind of Biodegradable nerve conduit structure and preparation method thereof of MULTILAYER COMPOSITE braiding |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5987138B2 (en) * | 2010-08-31 | 2016-09-07 | 国立研究開発法人農業・食品産業技術総合研究機構 | Cylindrical structure and manufacturing method thereof |
| US8877498B2 (en) * | 2010-12-01 | 2014-11-04 | Drexel University | Porous polymer scaffolds for neural tissue engineering and methods of producing the same |
| EP3490624B1 (en) * | 2016-08-01 | 2022-02-09 | Association for the Advancement of Tissue Engineering and Cell based Technologies & Therapies (A4TEC) - Associação | Nerve guidance conduits, methods of production and uses thereof |
| CN107982580A (en) * | 2017-12-08 | 2018-05-04 | 南通大学 | A kind of fibroin albumen tissue engineering bracket based on braiding and coating technology and its preparation method and application |
| CN110464881B (en) * | 2019-07-05 | 2021-09-14 | 武汉纺织大学 | Silk fibroin porous scaffold with hierarchical structure and preparation method thereof |
| CN111281599A (en) * | 2020-03-19 | 2020-06-16 | 中国海洋大学 | Enhanced artificial nerve conduit and preparation method and application thereof |
| CN112263714B (en) * | 2020-10-15 | 2022-06-07 | 苏州大学 | Silk protein nerve conduit and preparation method thereof |
-
2020
- 2020-10-15 CN CN202011102746.5A patent/CN112263714B/en active Active
-
2021
- 2021-04-09 WO PCT/CN2021/086167 patent/WO2022077878A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010036992A2 (en) * | 2008-09-26 | 2010-04-01 | Trustees Of Tufts College | Active silk muco-adhesives, silk electrogelation process, and devices |
| CN107952112A (en) * | 2017-12-07 | 2018-04-24 | 武汉纺织大学 | A kind of nerve trachea of the function of directional guide containing silk fibroin nano-fiber and preparation method thereof |
| CN108114315A (en) * | 2018-02-22 | 2018-06-05 | 南通大学 | A kind of mariages fibroin nerve graft |
| CN110251732A (en) * | 2019-06-18 | 2019-09-20 | 南通纺织丝绸产业技术研究院 | A kind of Biodegradable nerve conduit structure and preparation method thereof of MULTILAYER COMPOSITE braiding |
Non-Patent Citations (1)
| Title |
|---|
| 王丽丽: ""取向丝素蛋白纳米纤维凝胶的构建及其调控细胞行为的研究"", 《中国优秀博硕士学位论文全文数据库(博士) 基础科学辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022077878A1 (en) * | 2020-10-15 | 2022-04-21 | 苏州大学 | Silk protein nerve conduit and preparation method therefor |
| CN113180885A (en) * | 2021-04-01 | 2021-07-30 | 重庆市畜牧科学院 | Optic nerve interface and its preparing method |
| CN117018290A (en) * | 2023-10-10 | 2023-11-10 | 常州丝波敦生物科技有限公司 | Silk protein nanofiber nerve conduit with bionic orientation structure and preparation method thereof |
| CN117018290B (en) * | 2023-10-10 | 2024-01-05 | 常州丝波敦生物科技有限公司 | Silk protein nanofiber nerve conduit with bionic orientation structure and preparation method thereof |
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