CN101579246B - Artificial silk fibroin nano-fiber nerve repair conduit and preparation method thereof - Google Patents
Artificial silk fibroin nano-fiber nerve repair conduit and preparation method thereof Download PDFInfo
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- CN101579246B CN101579246B CN2009100338233A CN200910033823A CN101579246B CN 101579246 B CN101579246 B CN 101579246B CN 2009100338233 A CN2009100338233 A CN 2009100338233A CN 200910033823 A CN200910033823 A CN 200910033823A CN 101579246 B CN101579246 B CN 101579246B
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Abstract
The invention discloses a nerve repair conduit and a preparation method thereof. The nerve repair conduit consists of an inner layer conduit and an outer layer fiber wall, wherein the inner layer conduit consists of silk fibroin fibers arranged in the extending direction of the conduit, the outer layer fiber wall consists of the silk fibroin fibers encircling the conduit vertically and arranged axially, and the diameters of the silk fibroin fibers are between 50 and 3,000 nanometers; and the method for preparing the nerve repair conduit comprises the following steps: (1) preparing a regenerative silk fibroin film; (2) dissolving a pure regenerative silk fibroin film and preparing the silk fibroin fibers by using electrostatic spinning; and (3) collecting the silk fibroin fibers by using a roller device to obtain the nerve repair conduit. The nerve repair conduit is made from high-purity silk fibroin and is biodegradable, and the inner layer is made from parallel fibers arranged in the extending direction of the conduit and can conduct nerve cell migration; and the outer layer fibers are arranged by encircling the conduit axially and can support the conduit to prevent collapse.
Description
Technical field
The present invention relates to bridge material of a kind of peripheral nerve and neurologic defict and preparation method thereof.
Background technology
Because modern society produces and the continuous quickening of rhythm of life, the probability that people come to harm increases greatly.Because the damage that causes of accident, usually with the damage of peripheral nervous system.Nerve injury since the tissue can not be successful regeneration, often cause the long-term forfeiture of function.Treat comparatively serious peripheral nerve injury at present clinically, adopt the method for nerve autograft usually.The method repairing effect is better, is " goldstandard " of repairing peripheral nerve defection.But Method for autologous transplantation also has a lot of insufficient places, such as the restriction in the neural source of donor and the afunction of donor nerve etc.For overcoming these problems, adopting the artificial nerve catheter method to induce injured nerve regeneration is an ideal replacement method, also is the emphasis of studying in the present neural reparation field.The nerve trachea method is to use the catheterostat of synthetic between neural stump, and the two ends of conduit are entangled nearside and distally stump respectively, thus the neural regeneration of guiding.
Nerve trachea material commonly used at present mainly is divided into two classes: biological non-degradable and biodegradable material.In biological non-degradable material, silica gel at first is used to the artificial nerve catheter method and repairs neural.But silica gel long-term existence in vivo can produce challenges such as lasting inflammation, fibrosis and nerve compression, therefore often needs operation for the second time to remove conduit.Can overcome these shortcomings with Biodegradable material.The degradable nerve trachea material of research mainly is collagen, polylactic acid and polyglycolic acid etc. at present.Collagen has the affine performance of good nerve, and neurocyte is good in the collagen superficial growth.But the mechanical property of collagen is relatively poor, need to use cross-linking agent to improve its mechanical performance, and cross-linking agent of using usually such as glutaraldehyde etc. all has very high cytotoxicity, and after cross-linking agent was crosslinked, the biocompatibility of collagen can reduce.In addition, collagen protein has immunogenicity, can cause the immunological rejection of human body.These drawbacks limit collagen repair Application for Field at nerve.Polylactic acid and polyglycolic acid are the materials of synthetic, preparation technology's more complicated, and the price comparison costliness, and also the metabolite of these two kinds of materials is acidic materials, and human body is had stimulation, can influence the repairing effect of injured nerve.
Fibroin albumen is a kind of pure natural protein, has the body inside and outside biocompatibility equal with collagen protein, and can natural degradation, and the degraded end product is an aminoacid, is suitable as very much biomaterial.There are some researches show sticking, move and growing of fibroin albumen support neurocyte.After people such as Gu Xiaosong are dissolved fibroin albumen, the conduit of the eggshell structure of preparing by mould research experiment in body confirm to promote the mouse neurologic defect regeneration (referring to: Xiaosong GU et al.Biomaterials.28 (2007) 5526-5535), but, the migration of neurocyte there is not help because the inwall of the conduit of above-mentioned eggshell structure is a network structure.
Summary of the invention
The object of the invention provide a kind of avirulence, good biocompatibility, in vivo can natural degradation nerve rehabilitating tube, improve the migration of neurocyte.
For achieving the above object, the concrete technical scheme of the present invention is, a kind of nerve rehabilitating tube, the diameter of described nerve rehabilitating tube is 1.1～1.3 times of the diameter nerve that will repair, described nerve rehabilitating tube is made of inner conductor and outer layer fiber wall, described inner conductor is made of the fibroin fiber of arranging along conduit extension direction, the thickness of inner conductor is 1～200 μ m, described outer layer fiber wall is made of the fibroin fiber of arranging around inner conductor, the thickness of outer layer fiber wall is 0.01～1mm, and the diameter of described fibroin fiber is 50～3000nm;
In the technique scheme, the fibroin fiber that constitutes described inner conductor is that the extension direction (being the central axis direction of conduit) that is parallel to conduit is arranged in theory, but because the restriction of existing technology of preparing, in the practical situation, there is small angle between fibroin fiber and the catheter center's axle, can be similar to and thinks and be parallel to central axis direction; The fibroin fiber that constitutes described outer layer fiber wall is in theory perpendicular to the central axis direction of conduit, the cross section that promptly is parallel to conduit, same because the restriction of existing technology of preparing, in the practical situation, constitute between the cross section of the fibroin fiber of described outer layer fiber wall and conduit and have small angle, can be similar to and think perpendicular to central axis direction.
Further in the technical scheme, can contain the guiding fiber that helps cell adhesion and migration in the nerve rehabilitating tube, described guiding fiber is the natural fibroin fiber that comes unstuck;
Further in the technical scheme, come unstuck natural Bombyxmori Linnaeus, Antherea pernyi Guerin-Meneville or Semen Ricini silkworm fibroin fiber are set in the outer layer fiber wall.Thereby obtain the conduit of higher mechanical property;
Further in the technical scheme, can contain the seed cell of tool therapeutical effect in the inner conductor, described seed cell is selected from: one or more in neural stem cell, medulla mesenchyma cell, fibroblast, schwann cell or the Olfactory essheathing cell;
Further in the technical scheme, can contain the cytokine with therapeutical effect in the inner conductor, described cytokine is selected from one or more in nerve growth factor, ciliary nerves somatomedin, neurotrophic factor, brain-derived growth factor, basic fibroblast growth factor or the glial cell line-derived neurotrophic factor;
A kind of method for preparing above-mentioned nerve rehabilitating tube may further comprise the steps:
(1) silkworm silk is through coming unstuck, making pure regenerated silk fibroin film after dissolving, dialysis and the drying;
(2) use formic acid or hexafluoroisopropanol (HFIP) to dissolve pure regenerated silk fibroin film, use method of electrostatic spinning to prepare fibroin fiber;
(3) collect fibroin fiber by drum apparatus and obtain nerve rehabilitating tube;
Described silkworm silk is selected from: a kind of in mulberry silk, tussah silk or the ricinus silk;
In the technique scheme, the technology of silkworm silk being made regenerated silk fibroin film is a prior art, is published in the article " regeneration Bombyxmori Linnaeus fibroin/tussah silk fibroin static spins the research of nonwoven web structure " of the 23rd the 4th phase of volume of " polymer material science and engineering " July in 2007 referring to people such as Zuo Baoqi;
In the technique scheme, method of electrostatic spinning prepares fibroin fiber can reference: T B Bini, Shujun Gao, Ter Chyan Tan, ShuWang, Aymeric Lim, LimBen Hai and S Ramakrishna.NANOTECHNOLOGY. (2004) 1459-1464; Difference be with the regenerated silk fibroin among the present invention replace in the above-mentioned document polylactic acid-glycolic acid (Poly (L-lactide-co-glycolide), PLGA);
In the technique scheme, the parameter of method of electrostatic spinning is: spinning voltage is 0-50kv, and the spinning distance is 0-30cm, and spinning concentration is 0-15%, and spinning speed is 0-1ml/h; Drum rotation speed is at 0-10000rpm, and translational speed is 0-0.5m/s;
In the technique scheme, step (3) by the concrete steps that drum apparatus collection fibroin fiber obtains nerve rehabilitating tube is:
(1) drum apparatus is in low speed rotation high speed translation state, and fibroin fiber is wrapped in the inner conductor that the drum apparatus surface forms nerve rehabilitating tube along with drum apparatus moves along catheter shaft to sinking;
(2) reduce the point-to-point speed of drum apparatus, heighten rotary speed, fibroin fiber influences the lower edge catheter drive to being arranged in parallel the high speed rotating of drum apparatus, and the surface that is wrapped in inner conductor forms the outer layer fiber wall of nerve rehabilitating tube;
Rotating speed≤the 25r/min of described low speed rotation, the rotating speed 〉=1500r/min of high speed rotating; Speed≤the 2.5cm/s of described low speed translation, the speed 〉=8cm/s of high speed translation;
The above-mentioned principle for preparing the method for nerve rehabilitating tube is: the spray webbing syringe needle of electrostatic spinning is arranged on the top (reference: T B Bini of drum apparatus, Shujun Gao, Ter Chyan Tan, ShuWang, Aymeric Lim, LimBen Hai and S Ramakrishna.NANOTECHNOLOGY. (2004) 1459-1464), the drum apparatus among the present invention is that the rotatable while of drum apparatus can be along translation before and after the direction of central shaft with the central shaft; At the spinning initial stage, allow the rotary speed of drum apparatus be in lower-speed state, the high speed translation, fiber just can be by the rotation of drum apparatus influence like this, and moving along catheter shaft to sinking, after the spinning certain thickness along with drum apparatus just; Reduce point-to-point speed, heighten rotary speed, fiber can not be subjected to the influence of drum apparatus translation like this, and be the high speed rotating of drum apparatus influence the lower edge catheter drive to being arranged in parallel, spinning needs thickness.
Further in the technical scheme, inject the fibroin aqueous solution in the nerve rehabilitating tube;
Further in the technical scheme, the natural mulberry silk fibroin fiber that will come unstuck places the outer layer fiber wall;
Further in the technical scheme, improve the mechanical property of conduit again at skin spinning one deck polymeric biomaterial of outer fibre wall, described polymeric biomaterial can be selected from but be not limited to polylactic acid (PLA), thereby forms the hollow artificial mulberry silk fibroin of high strength/PLA composite nano fiber nerve rehabilitating tube;
Further in the technical scheme, the seed cell that contains the tool therapeutical effect can be injected nerve rehabilitating tube, described seed cell is selected from: one or more in neural stem cell, medulla mesenchyma cell, fibroblast, schwann cell or the Olfactory essheathing cell;
Further in the technical scheme, can inject nerve rehabilitating tube with containing the cytokine with therapeutical effect, described cytokine is selected from one or more in nerve growth factor, ciliary nerves somatomedin, neurotrophic factor, brain-derived growth factor, basic fibroblast growth factor or the glial cell line-derived neurotrophic factor;
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
(1) nerve rehabilitating tube material therefor of the present invention is the high-purity silkworm fibroin, and this protein can be degraded under field conditions (factors), and the degraded end product is aminoacid, and is harmless;
(2) the described nerve rehabilitating tube of this product is a hollow tubing conductor, is divided into inside and outside two-layerly, and internal layer is parallel fiber vertically, can play the effect that guides neuranagenesis; Skin is around the conduit axially-aligned, plays supporting tube and prevents the effect of subsiding;
(3) because this product is made up of nanofiber, so will gappedly exist between fiber, the existence in this gap can allow nutrient substance pass through smoothly, and provides space and nutrition for nerve growth.
Description of drawings
The sem photograph that is parallel to the axial fiber of nerve rehabilitating tube among Fig. 1 embodiment four;
Nerve rehabilitating tube longitudinal profile sem photograph among Fig. 2 embodiment four;
The profile scanning Electronic Speculum figure of nerve rehabilitating tube inner conductor among Fig. 3 embodiment four;
The profile scanning Electronic Speculum figure of nerve rehabilitating tube outer layer fiber wall among Fig. 4 embodiment four.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment one
(1) natural mulberry silk boils 30min with the sodium carbonate liquor of mass fraction 0.05% and comes unstuck, and obtains pure mulberry silk fibroin fiber after repeating 3 times.This fibroin albumen is dissolved in LiBr: C
2H
5OH=40: stirring and dissolving acquisition regenerated silk fibroin water solution in 80 ± 2 ℃ of water-baths of constant temperature in 60 (mass ratioes).Become regenerated silk fibroin film after the dialysis drying.
(2) prepared regenerated silk fibroin film in the step (1) is dissolved in the formic acid, makes the regenerated silk fibroin artificial neuron by electrospinning process and repair conduit.
(3) in step (2) rotation by drum apparatus with move obtain the internal layer parallel conduits axially, outer layer ring is around the conduit of catheter shaft to arrangement architecture, concrete grammar is: at first, the rotary speed of drum apparatus is in lower-speed state (20r/min), high speed translation 12cm/s forms parallel fiber and constitutes inner conduit (200 microns of thickness); Reduce point-to-point speed (2cm/s), heighten rotary speed (2000r/min), form outer layer fiber wall (200 microns of thickness);
(4) in step (3), inject the fibroin aqueous solution in the obtained nerve rehabilitating tube, after the lyophilization be artificial silk fibroin nano-fiber nerve repair conduit.
Embodiment two:
(1) the natural tussah silk mass fraction is that 5% sodium carbonate liquor boils the 30min that comes unstuck, and obtains pure tussah silk fibroin fiber after repeating 3 times.
(2) prepared tussah silk peptide fiber in the step (1) is dissolved in the lithium rhodanate solution.Deionized water dialysis 3 days becomes regeneration tussah silk fibroin film after the drying.
(3) prepared regeneration tussah silk fibroin film in the step (2) is dissolved among the HFIP, make regeneration tussah silk fibroin artificial neuron by electrospinning process and repair conduit: at first, the rotary speed 10r/min of drum apparatus, point-to-point speed 15cm/s forms parallel fiber and constitutes inner conduit (300 microns of thickness); Adjust point-to-point speed 2cm/s, rotary speed 2200r/min forms outer layer fiber wall (300 microns of thickness).
(4) in the conduit spinning process of step (3), place the outer layer fiber wall to obtain the conduit of higher mechanical property by the natural mulberry silk fibroin fiber that will come unstuck.
Embodiment three:
(1) natural mulberry silk boils 30min with the sodium carbonate liquor of mass fraction 0.05% and comes unstuck, and obtains pure mulberry silk fibroin fiber after repeating 3 times.This fibroin fiber is dissolved in LiBr: C
2H
5OH=40: stirring and dissolving acquisition regenerated silk fibroin water solution in 80 ± 2 ℃ of water-baths of constant temperature in 60 (mass ratioes).Become regenerated silk fibroin film after the dialysis drying.
(2) prepared regenerated silk fibroin film in the step (1) is dissolved in the formic acid, makes the regenerated silk fibroin artificial neuron by electrospinning process and repair conduit.
(3) in step (2) rotation by collecting pipe with move obtain the internal layer parallel conduits axially, outer layer fiber vertical conduit threaded shaft is to structure arranged (with embodiment one).
(4) obtained conduit is before the demoulding in the step (3), and the system of respinning one deck polymeric biomaterial improves the mechanical property of conduit, and polymeric biomaterial is PLA.Form the hollow artificial mulberry silk fibroin of high strength/PLA composite nano fiber nerve rehabilitating tube.
Embodiment four:
(1) the natural tussah silk mass fraction is that 5% sodium carbonate liquor boils the 30min that comes unstuck, and obtains pure tussah silk fibroin fiber after repeating 3 times.
(2) prepared tussah silk peptide fiber in the step (1) is dissolved in the lithium rhodanate solution.Deionized water dialysis 3 days becomes regeneration tussah silk fibroin film after the drying.
(3) prepared regeneration tussah silk fibroin film in the step (2) is dissolved among the HFIP, makes regeneration tussah silk fibroin artificial neuron by electrospinning process and repair conduit (with embodiment two).
This nerve rehabilitating tube is carried out the electron-microscope scanning analysis;
(4) schwann cell is injected in the conduit prepared in the step (3), forms artificial tussah silk fibroin/seed cell composite nerve and repair conduit.
Claims (8)
1. nerve rehabilitating tube, the diameter of described nerve rehabilitating tube is 1.1～1.3 times of the diameter nerve that will repair, it is characterized in that: described nerve rehabilitating tube is made of inner conductor and outer layer fiber wall, described inner conductor is made of the fibroin fiber of arranging along conduit extension direction, and described outer layer fiber wall is made of the fibroin fiber of arranging around inner conductor.
2. nerve rehabilitating tube according to claim 1 is characterized in that: the thickness of described inner conductor is 1～200 μ m, and the thickness of outer layer fiber wall is 0.01～1mm, and the diameter of described fibroin fiber is 50～3000nm.
3. nerve rehabilitating tube according to claim 1 is characterized in that: contain the guiding fiber in the nerve rehabilitating tube, described guiding fiber is the natural fibroin fiber that comes unstuck.
4. nerve rehabilitating tube according to claim 1 is characterized in that: come unstuck natural Bombyxmori Linnaeus, Antherea pernyi Guerin-Meneville or Semen Ricini silkworm fibroin fiber are set in the outer layer fiber wall.
5. nerve rehabilitating tube according to claim 1, it is characterized in that: contain the seed cell of tool therapeutical effect in the inner conductor, described seed cell is selected from: one or more in neural stem cell, medulla mesenchyma cell, fibroblast, schwann cell or the Olfactory essheathing cell.
6. nerve rehabilitating tube according to claim 1, it is characterized in that: contain the cytokine with therapeutical effect in the inner conductor, described cytokine is selected from one or more in nerve growth factor, brain-derived growth factor or the basic fibroblast growth factor.
7. method for preparing the described nerve rehabilitating tube of claim 1 is characterized in that: may further comprise the steps:
(1) silkworm silk is through coming unstuck, making pure regenerated silk fibroin film after dissolving, dialysis and the drying;
(2) use formic acid or hexafluoroisopropanol to dissolve pure regenerated silk fibroin film, use method of electrostatic spinning to prepare fibroin fiber;
(3) collect fibroin fiber by drum apparatus and obtain nerve rehabilitating tube;
Described silkworm silk is selected from: a kind of in mulberry silk, tussah silk or the ricinus silk.
8. preparation method according to claim 7 is characterized in that: step (3) by the concrete steps that drum apparatus collection fibroin fiber obtains nerve rehabilitating tube is:
(1) drum apparatus is in low speed rotation high speed translation state, and fibroin fiber is wrapped in the inner conductor that the drum apparatus surface forms nerve rehabilitating tube along with drum apparatus moves along catheter shaft to sinking;
(2) point-to-point speed of reduction drum apparatus, heighten rotary speed, make drum apparatus be in high speed rotating low speed translation state, fibroin fiber influences the lower edge catheter drive to being arranged in parallel the high speed rotating of drum apparatus, and the surface that is wrapped in inner conductor forms the outer layer fiber wall of nerve rehabilitating tube;
Rotating speed≤the 25r/min of described low speed rotation, the rotating speed 〉=1500r/min of high speed rotating; Speed≤the 2.5cm/s of described low speed translation, the speed 〉=8cm/s of high speed translation.
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| CN101912318B (en) * | 2010-07-30 | 2012-11-07 | 东华大学 | Three-layer electrostatic spinning ordered fiber nerve conduit and preparation and application thereof |
| US8758374B2 (en) | 2010-09-15 | 2014-06-24 | University Of Utah Research Foundation | Method for connecting nerves via a side-to-side epineurial window using artificial conduits |
| CN102048595B (en) * | 2010-12-21 | 2013-05-15 | 东华大学 | Degradable nerve conduit with highly-oriented tube-in-tube structure and manufacturing method thereof |
| US10842494B2 (en) | 2011-10-17 | 2020-11-24 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| US9931121B2 (en) | 2011-10-17 | 2018-04-03 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| CN105983135A (en) * | 2015-01-28 | 2016-10-05 | 五石医疗科技（苏州）有限公司 | Developing-able neural restoration conduit and preparation method thereof |
| CN105983136A (en) * | 2015-01-28 | 2016-10-05 | 五石医疗科技（苏州）有限公司 | Neural restoration catheter and preparation method for same |
| CN105233344B (en) * | 2015-10-22 | 2018-04-03 | 温州医科大学 | A kind of composite repairing material and its support for being used to bridge defect nerve |
| CN105748171B (en) * | 2016-02-21 | 2017-09-15 | 新乡医学院 | Biological nerve duct |
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| CN106983915B (en) * | 2017-05-24 | 2019-09-06 | 南通市第一人民医院 | Silk microfibre-gelatin-polyethylene glycol medical catheter and preparation method thereof |
| EP3427764A1 (en) * | 2017-07-12 | 2019-01-16 | Université de Technologie de Compiègne | Fibrous polymer material comprising fibroin and polymer scaffolds comprising thereof |
| CN109172864A (en) * | 2018-10-10 | 2019-01-11 | 上海市第人民医院 | A kind of schwann cell combines LC-PLGA conduit and its application with neural stem cell |
| CN110038166B (en) * | 2019-04-24 | 2021-06-22 | 温州医科大学 | Self-curling film agent applied to nerve repair and preparation method thereof |
| CN110251732B (en) * | 2019-06-18 | 2020-10-27 | 南通纺织丝绸产业技术研究院 | Multilayer composite braided degradable nerve conduit structure and preparation method thereof |
| CN111895902B (en) * | 2020-07-20 | 2022-04-22 | 苏州大学 | Ultrathin transparent carbon nanofiber membrane flexible strain sensor and preparation method thereof |
| CN113180885A (en) * | 2021-04-01 | 2021-07-30 | 重庆市畜牧科学院 | Optic nerve interface and its preparing method |
| CN114732956A (en) * | 2022-04-20 | 2022-07-12 | 西南大学 | Preparation method of nerve scaffold with high matching degree of in-vivo degradation and nerve regeneration |
| 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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