CN104645412B - A kind of preparation method of the biomimetic prosthetic Nerve Scaffold built by collagen protein - Google Patents
A kind of preparation method of the biomimetic prosthetic Nerve Scaffold built by collagen protein Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of biomimetic prosthetic Nerve Scaffold built by collagen protein.The method is standby for collagen protein is first made following three different shape: 1) is dissolved with hexafluoroisopropanol, uses electrostatic spinning technique to make nano-fiber film after drying by protein frozen；2) combine lyophilization and mechanical stretching technology and albumen is prepared as including the line style support of orderly trickle pipeline；3) collagen is dissolved in pure water it is prepared as hydrogel；Then, nano-fiber film is rolled into tubulose simulation epineurium, orderly pipe line type scaffold mimics perineurium is filled in pipe, fill artificial neuron support prepared by the hydrogel analog neuron inner membrance present invention in the pipeline of final online type support and in standoff gap and preferably simulate perineural supporting structure based on bionics theory, on the basis of ensureing good mechanical property, can promote and guide aixs cylinder and Scs to grow in an orderly manner and by the peripheral nerve of defect.
Description
Technical field
The invention belongs to the preparation field of tissue engineering artificial Nerve Scaffold, particularly to the preparation method of a kind of biomimetic prosthetic Nerve Scaffold built by collagen protein.
Background technology
nullDevelopment along with modern society,Accident injury war earthquake tumor operation etc. causes the number of nerve injury to increase year by year,In the whole world increases wound case every year newly,Peripheral nerve injury case accounts for 1.5% ~ 4.0% China's peripheral nerve injury case and increases 60 ~ 900,000 examples every year newly,The case being repaired nerve by neural transplantation is wherein needed to be about 300,000 ~ 450,000 examples,If malpractice causes life-long disabilities,The reparation causing serious commercial burden and burden on society peripheral nerve defection is that a great problem of current wound surgery microsurgery is along with the development of tissue engineering with reconstruction,Peripheral nerve tissue's engineering is the most day by day carried out,Being acknowledged as one of effective means solving this problem nerve trachea is that many scholars propose on the basis of summing up forefathers' research: it is the pipeline sampling device that a kind of natural or synthetic material are made,It is used for guiding the neural of neurologic defect near-end to extend regeneration,Connect neurologic defect two ends,And reduce and around become the neurotrophic factor isoreactivity material of the infiltration of fibrous tissue and the secretion of allowable damage nervous tissue to promote neuranagenesis in diffusing into conduit.
Oneself finds multiple neural substitution material up to now, including biomaterial such as: autologous tissue's peripheral heart amniotic duct;Allosome tissue's nervus vasculairs etc., achieved with certain repairing effect but owing to these materials easily cause the immunological rejection of host or easily subside, draw materials and purify and more complicated, and can not the most accurately control the length of conduit, diameter, the physical behaviors such as pipe thickness, limit its clinical practice current, to have plasticity strong because of it for what both at home and abroad research was more is Biodegradable material wherein collagen protein, immunological rejection is low, the advantages such as acquisition is convenient receive and are increasingly widely applied still, at present the collagen-based materials of report is due to the reason such as lack of alignment of pore size and collagen fiber, can't well guide the growth of regenerating nerve.
Summary of the invention
For the problem solving above-mentioned existence, the collagen-based materials that the present invention uses lyophilization pulling method to make has the hole close with nerve tract, and collagen fiber are ordered arrangement, can analog neuron bundle film in periphery, with the bigger collagen nanofiber thin-film material of hardness as support, can ensure that artificial neuron has higher mechanical strength based on above advantage, this artificial neural material has bigger application prospect in the clinical practice of neuranagenesis reparation
It is an object of the invention to provide the preparation method of a kind of biomimetic prosthetic Nerve Scaffold built by collagen protein.
The technical solution used in the present invention is:
The preparation method of a kind of biomimetic prosthetic Nerve Scaffold built by collagen protein, comprises the following steps:
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 15～25mg/ml, pours into after mould in-25～-15 DEG C of freezings 60～80min, lyophilizing 15 ~ 25min under the conditions of-80 ~-60 DEG C of vacuum；Take out to obtain collagen-based materials；
Above-mentioned collagen-based materials is stretched to 1.5 ~ 2.5 times of raw footage, the most in a stretched state, it is placed on-25～-15 DEG C of freezings 60～80min, secondary freeze drying 1.5 ~ 2.5h is carried out under the conditions of placing into-80 ~-60 DEG C of vacuum, obtain the internal line style support containing orderly trickle pipeline, the most orderly collagen scaffold, standby；
The preparation of nano-fiber film: the hexafluoroisopropanol solution of preparation collagen protein powder, makes nano-fiber film by this solution, standby；
The preparation of collagen hydrogel: prepare collagen hydrogel；Standby；
2) above-mentioned nano-fiber film is rolled into tubulose with analog neuron adventitia, then the orderly collagen scaffold of above-mentioned preparation will be filled in nano-fiber film pipe with analog neuron bundle film, finally with all gaps in collagen hydrogel filling pipe with analog neuron inner membrance, biomimetic prosthetic Nerve Scaffold can be obtained.
Further, above-mentioned steps 1) described in the column collagen-based materials that collagen-based materials is long 0.8 ~ 1.2cm, diameter 0.8 ~ 1.2mm.
Further, in above-mentioned steps 1) in, with the speed of 1 ~ 1.4mm/s, collagen-based materials is stretched to 1.5 ~ 2.5 times of raw footage with drawing device.
Further, above-mentioned steps 1) described in the concentration of hexafluoroisopropanol solution of collagen protein powder be 80～120mg/ml.
Further, above-mentioned steps 1) in, use electrostatic spinning technique that the hexafluoroisopropanol solution of collagen protein powder is made the nano-fiber film that thickness is 1.8～2.2mm.
Further, above-mentioned steps 1) in, prepare the method for collagen hydrogel be compound concentration be the collagenic aqueous solution of 0.8～1.2mg/ml, collagen hydrogel can be obtained.
Further, above-mentioned steps 2) in, nano-fiber film is rolled into a diameter of 1 ~ 2mm, the tubulose of a length of 1 ~ 1.5cm.
Further, above-mentioned steps 2) in, fill orderly collagen scaffold by microscope microforceps by nano-fiber film pipe.
The invention has the beneficial effects as follows:
1) the artificial neuron support that prepared by the present invention preferably simulates perineural supporting structure based on bionics theory, on the basis of ensureing good mechanical property, can promote and guide aixs cylinder and Scs to grow in an orderly manner and by the peripheral nerve of defect.
2) present invention utilizes electrospinning, freezer dryer and pulling force to make collagen protein form the support of specific pore, the three dimensional structure of normal neuronal can be simulated, fiber alignment linearly contributes to guiding the growth of nerve tract simultaneously, incorporating collagen itself has the advantages that degradable immunological rejection is low, constructs a kind of excellent neuranagenesis timbering material
3) in the present invention, the structure that collagen fiber are formed in ordered arrangement, contributes to guiding the oriented growth of neural axon under the effect of pulling force；In periphery, with the bigger collagen nanofiber thin-film material of hardness as support, it is ensured that artificial neuron has higher mechanical strength；Finally fill hydrogel at gap portion, construct a kind of bionical nerve repair material close to normal neuronal.
Accompanying drawing explanation
Fig. 1 is the stretching schematic diagram of collagen-based materials in the present invention；
Fig. 2 is the collagen-based materials of the present invention tool in drawing process situation of change (A～C) originally；
Fig. 3 is outer surface situation (A and B) and the visual condition (C) of orderly collagen scaffold of the biomimetic prosthetic Nerve Scaffold of embodiment 1 preparation；
Fig. 4 is the scanning electron microscope (SEM) photograph of the orderly collagen scaffold longitudinal section structure of embodiment 1 preparation；
Fig. 5 is the cross section scanning electron microscope (SEM) photograph (A) of the collagen scaffold that embodiment 1 prepares the scanning electron microscope (SEM) photograph (B) of the internal Structure of cross section of biomimetic prosthetic Nerve Scaffold and prepared by conventional method；
Fig. 6 is the structural representation of biomimetic prosthetic Nerve Scaffold of the present invention；
Fig. 7 is the axon regeneration effect that rat nerve injury is repaired, and A is that positive controls nerve autograft does the result repaired；B is to do, with the biomimetic prosthetic Nerve Scaffold of this bright preparation, the result repaired, and C figure is the result of negative control group.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further it should be understood that, these embodiments be merely to illustrate the present invention rather than limit the scope of the present invention it should also be understood that, after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally
Embodiment
1
1) prepare the collagen solution (being made into by commercial collagen protein powder and water) that concentration is 20mg/ml, pour into after mould in-20 DEG C of freezing 1h, place into-70 DEG C of lyophilizing 20min of vacuum in freezer dryer；Take out to obtain the column collagen-based materials of long 1cm diameter 1mm；
2) above-mentioned collagen-based materials drawing device (for market existing equipment, be can be selected for U.S. Sutter
The program control horizontal microelectrode of InstrumentP97 type draws instrument) with the twice (stretching schematic diagram is as shown in Figure 1) of the speed slow stretching of 1.2mm each second to raw footage, the collagen-based materials concrete change procedure in drawing process is as shown in Fig. 2 A～C, then together with equipment at-20 DEG C of freezing 1h, it is placed in-70 DEG C of secondary freeze drying 2h of vacuum again, obtain the internal line style support (collagen scaffold i.e. in order, as shown in Fig. 3 C and Fig. 4) containing orderly trickle pipeline；Standby；
3) being dissolved by collagen protein powder (commercial) hexafluoroisopropanol, in gained solution, collagen concentration is 100mg/ml, and this solution uses electrostatic spinning technique to make the nano-fiber film that thickness is 2mm；Standby；
4) prepare the collagenic aqueous solution that concentration is 1mg/ml, obtain collagen hydrogel；Standby；
5) above-mentioned nano-fiber film is held diameter 1.5mm polyethylene mould and be rolled into a diameter of 1.5mm, the tubulose of a length of 1.2cm, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (collagen scaffold i.e. in order) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of the above-mentioned preparation of filling in pipe, fill full collagen hydrogel with analog neuron inner membrance with microsyringe in the pipeline of final online type support (i.e. in order collagen scaffold) and in all gaps such as standoff gap, biomimetic prosthetic Nerve Scaffold can be obtained.
Biomimetic prosthetic Nerve Scaffold prepared by the present embodiment as shown in Figure 3 A and Figure 3 B, it can be seen that tube-surface is coarse, has more uniform space to can ensure that the diffusive transport of nutrient substance and metabolic waste.In the present embodiment, outward appearance is as shown in institute Fig. 3 C for the line style support (collagen scaffold i.e. in order) containing orderly trickle pipeline of preparation, it can be seen that a certain degree of linear structure of its surface display.
The longitudinal section structure of the line style support (collagen scaffold i.e. in order) of the orderly trickle pipeline prepared by the present embodiment is scanned Electronic Speculum detection, testing result is as shown in Figure 4, it may be seen that its internal structure forms linear halfpipe, the most substantially hinder grow to walk crosswise partition at long axis direction.
The internal Structure of cross section of the biomimetic prosthetic Nerve Scaffold prepared by the present embodiment is scanned Electronic Speculum detection, testing result is as shown in Figure 5 B, it can be seen that cross section is in the hole differed in size, pore diameter is 20-80 μm, illustrate that tubular structure is obvious, its porosity is close with natural nerve tract, can well simulate its structure, promotes the growth of aixs cylinder.And Fig. 5 A is collagen scaffold prepared by existing conventional method, not having stretched, its internal structure is laminated structure at random；The concrete operations of the most existing conventional method can refer to di Summa PG, Kingham
PJ,et al.Collagen (NeuraGen®) nerve conduits and stem cells for
peripheral nerve gap repair.Neurosci Lett. 2014 Jun 20;572:26-31. or Cui
T, Yan Y, et al. Rapid Prototyping of a Double-Layer Polyurethane–Collagen Conduit for Peripheral Nerve Regeneration.Tissue
Eng Part C Methods. 2009 Mar;15 (1): 1-9. or Sirivisoot S, Pareta R,et al.Protocol and cell responses in three-dimensional
conductive collagen gel scaffolds with conductive polymer nanofibres
for tissue regeneration. Interface Focus. 2014 Feb 6;4(1):20130050.
As shown in Figure 6, outermost layer is the nano-fiber film (can be described as electro spinning nano fiber thin film) that electrostatic spinning technique is made to the structural representation of biomimetic prosthetic Nerve Scaffold of the present invention, includes orderly collagen scaffold and collagen gel inside nano-fiber film
Embodiment
2
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 25mg/ml, pours into after mould in-15 DEG C of freezing 80min, places into-60 DEG C of lyophilizing 25min of vacuum in freezer dryer；Take out to obtain the column collagen-based materials of long 1.2cm diameter 1.2mm；
2) by above-mentioned collagen-based materials drawing device with 2.5 times of the speed slow stretching of 1.4mm each second to raw footage, then at extended state, it is placed on-15 DEG C of freezing 80min, place into-80 DEG C of vacuum and carry out secondary freeze drying 1.5h, obtain the internal line style support containing orderly trickle pipeline, the most orderly collagen scaffold, standby；
3) preparation of nano-fiber film: being dissolved by collagen protein powder hexafluoroisopropanol, in gained solution, collagen concentration is 80mg/ml, this solution uses electrostatic spinning technique to make the nano-fiber film that thickness is 2.2mm；Standby；
4) preparation of collagen hydrogel: preparation concentration is the collagenic aqueous solution of 0.8mg/ml, obtains collagen hydrogel；Standby；
5) above-mentioned nano-fiber film is held diameter 2mm polyethylene mould and be rolled into a diameter of 2mm, a length of 1cm tubulose, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (collagen scaffold i.e. in order) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of the above-mentioned preparation of filling in pipe, last in the pipeline of orderly collagen scaffold and in gap, fill full collagen hydrogel with analog neuron inner membrance with microsyringe, biomimetic prosthetic Nerve Scaffold can be obtained.
Embodiment
3
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 15mg/ml, pours into after mould in-25 DEG C of freezing 60min, places into-80 DEG C of lyophilizing 15min of vacuum in freezer dryer；Take out to obtain the column collagen-based materials of long 0.8cm diameter 0.8mm；
2) by above-mentioned collagen-based materials drawing device with 1.5 times of the speed slow stretching of 1mm each second to raw footage, then at extended state, it is placed on-25 DEG C of freezing 60min, place into-60 DEG C of vacuum and carry out secondary freeze drying 2.5h, obtain the internal line style support containing orderly trickle pipeline, the most orderly collagen scaffold, standby；
3) preparation of nano-fiber film: being dissolved by collagen protein powder hexafluoroisopropanol, in gained solution, collagen concentration is 120mg/ml, this solution uses electrostatic spinning technique to make the nano-fiber film that thickness is 1.8mm；Standby；
4) preparation of collagen hydrogel: preparation concentration is the collagenic aqueous solution of 1.2mg/ml, obtains collagen hydrogel；Standby；
5) above-mentioned nano-fiber film is held diameter 1mm polyethylene mould and be rolled into a length of 1.5cm, the tubulose of a diameter of 1mm, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (collagen scaffold i.e. in order) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of the above-mentioned preparation of filling in pipe, last in the pipeline of orderly collagen scaffold and in gap, fill full collagen hydrogel with analog neuron inner membrance with microsyringe, biomimetic prosthetic Nerve Scaffold can be obtained.
The biomimetic prosthetic Nerve Scaffold prepared above-described embodiment below makees further effect detection.
The axon regeneration experiment that rat nerve injury is repaired
The biomimetic prosthetic Nerve Scaffold prepared by above-described embodiment 1 is used for the axon regeneration that rat nerve injury is repaired, rat right sciatic nerves is done 1cm defect, experimental group this biomimetic prosthetic Nerve Scaffold bridge joint is repaired, negative control group uses the empty set pipe that simple collagen rolls, and positive controls uses the goldstandard nerve autograft of nerve injury treatment.All repair materials all bridge defect by micro suture.The operation later moon, take graft stage casing and do rip cutting.Repairing effect is evaluated by the situation of detection neural axon regeneration.
Testing result is as it is shown in fig. 7, there it can be seen that A figure is to make the result repaired in positive controls of nerve autograft, the effect that its aixs cylinder (in figure shown in shiny red fluorescence) regenerates is best, and aixs cylinder density is big and linear arrangement is preferable.B figure is to do, with the biomimetic prosthetic Nerve Scaffold of this bright preparation, the result repaired, and the situation that aixs cylinder (in figure shown in shiny red fluorescence) regenerates is also preferably close to the effect of positive controls, and the situation of aixs cylinder linear growth is the most obvious.C figure is the result of negative control group, the most far short of what is expected, is hardly visible shiny red fluorescence in figure, almost without the regeneration of aixs cylinder.The above results illustrate biomimetic prosthetic Nerve Scaffold prepared by the inventive method can each Rotating fields of preferable analog neuron, promote the regeneration of neural axon in nerve injury.
Easy to understand for those skilled in the art; the foregoing is only the preferred embodiment of patent of the present invention; not in order to limit the present invention, any amendment, equivalent and the improvement etc. made within all the spirit and principles in the present invention, within all falling within the protection domain of application claims.
Claims (8)
1. the preparation method of the biomimetic prosthetic Nerve Scaffold that a kind is built by collagen protein, it is characterised in that: comprise the following steps:
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 15～25mg/ml, pours into after mould in-25～-15 DEG C of freezings 60～80min, lyophilizing 15 ~ 25min under the conditions of-80 ~-60 DEG C of vacuum；Take out to obtain collagen-based materials；
Above-mentioned collagen-based materials is stretched to 1.5 ~ 2.5 times of raw footage, the most in a stretched state, it is placed on-25～-15 DEG C of freezings 60～80min, secondary freeze drying 1.5 ~ 2.5h is carried out under the conditions of placing into-80 ~-60 DEG C of vacuum, obtain the internal line style support containing orderly trickle pipeline, the most orderly collagen scaffold, standby；
The preparation of nano-fiber film: the hexafluoroisopropanol solution of preparation collagen protein powder, makes nano-fiber film by this solution, standby；
The preparation of collagen hydrogel: prepare collagen hydrogel；Standby；
2) above-mentioned nano-fiber film is rolled into tubulose with analog neuron adventitia, then the orderly collagen scaffold of above-mentioned preparation will be filled in nano-fiber film pipe with analog neuron bundle film, finally with all gaps in collagen hydrogel filling pipe with analog neuron inner membrance, biomimetic prosthetic Nerve Scaffold can be obtained.
Preparation method the most according to claim 1, it is characterised in that: the collagen-based materials described in step 1) is the column collagen-based materials of long 0.8 ~ 1.2cm, diameter 0.8 ~ 1.2mm.
Preparation method the most according to claim 1, it is characterised in that: in step 1), with the speed of 1 ~ 1.4mm/s, collagen-based materials is stretched to drawing device 1.5 ~ 2.5 times of raw footage.
Preparation method the most according to claim 1, it is characterised in that: the concentration of the hexafluoroisopropanol solution of collagen protein powder described in step 1) is 80～120mg/ml.
Preparation method the most according to claim 1, it is characterised in that: in step 1), use electrostatic spinning technique that the hexafluoroisopropanol solution of collagen protein powder is made the nano-fiber film that thickness is 1.8～2.2mm.
Preparation method the most according to claim 1, it is characterised in that: in step 1), prepare the method for collagen hydrogel be compound concentration be the collagenic aqueous solution of 0.8～1.2mg/ml, collagen hydrogel can be obtained.
Preparation method the most according to claim 1, it is characterised in that: in step 2) in, nano-fiber film is rolled into a diameter of 1 ~ 2mm, the tubulose of a length of 1 ~ 1.5cm.
Preparation method the most according to claim 1, it is characterised in that: in step 2) in, fill orderly collagen scaffold by microscope microforceps by nano-fiber film pipe.
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| CN108452377A (en) * | 2017-02-21 | 2018-08-28 | 北京中科再康生物技术有限公司 | Growth factor composite collagen timbering material and function collagen as tissue engineering scaffold for repairing nerve damage |
| CN106806941B (en) * | 2017-02-22 | 2022-08-12 | 中国人民解放军第四军医大学 | Preparation method and application of easy-to-suture array microtubule nerve repair scaffold |
| CN107151646B (en) * | 2017-05-18 | 2021-07-09 | 西安交通大学 | Active biological battery construction method based on generating cells |
| CN108939165A (en) * | 2018-06-22 | 2018-12-07 | 中山大学附属第医院 | A kind of peripheral nerve bracket and preparation method thereof |
| CN108853583A (en) * | 2018-06-22 | 2018-11-23 | 中山大学附属第医院 | A kind of peripheral nerve bracket and preparation method thereof based on 3D printing |
| CN109172036B (en) * | 2018-06-22 | 2020-12-22 | 中山大学附属第一医院 | Multichannel peripheral nerve conduit and preparation method thereof |
| CN111214651A (en) * | 2019-12-05 | 2020-06-02 | 中山大学附属第一医院 | Application of hexa-type collagen in preparation of related medicines and transplants capable of improving order of regenerated nerves |
| CN111068118B (en) * | 2019-12-20 | 2021-12-07 | 中山大学 | Artificial nerve graft and preparation method thereof |
| CN114259601B (en) * | 2021-12-27 | 2023-05-26 | 暨南大学 | Living cell bionic cornea anterior lamina and preparation method thereof |
| CN114618017B (en) * | 2022-01-27 | 2023-06-02 | 华东理工大学 | Collagen film with highly oriented and crystalline collagen fiber structure and preparation method thereof |
| CN115634317B (en) * | 2022-10-20 | 2023-09-08 | 上海市第六人民医院 | Collagen fiber composite membrane for nerve injury repair |
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| TW528600B (en) * | 1996-11-20 | 2003-04-21 | Yasuhiko Shimizu | Artificial neural canal |
| WO1999063908A1 (en) * | 1998-06-10 | 1999-12-16 | Tapic International Co., Ltd. | Artificial neural tube |
| WO2005046457A2 (en) * | 2003-11-05 | 2005-05-26 | Texas Scottish Rite Hospital For Children | A biomimetic biosynthetic nerve implant |
| JP4605985B2 (en) * | 2002-12-27 | 2011-01-05 | ニプロ株式会社 | Nerve regeneration induction tube |
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