CN104645412A - Preparation method of bionic artificial nerve scaffold established by collagen - Google Patents
Preparation method of bionic artificial nerve scaffold established by collagen Download PDFInfo
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Abstract
The invention discloses a preparation method of a bionic artificial nerve scaffold established by a collagen. The method comprises the following steps: firstly preparing the collagen in three different shapes for later use: 1) dissolving the frozen and dried collagen by using hexafluoroisopropanol and preparing a nanofiber thin film by using an electrospinning technique; 2) preparing a linear scaffold containing an ordered fine pipeline by the collagen by combining freezing and drying with a mechanical stretching technology; and 3) dissolving the collagen in pure water to prepare a hydrogel; then, coiling the nanofiber thin film into a tube to simulate perilemma, wherein the ordered pipeline linear scaffold is filled in the tube to simulate perineurium; and finally, filling the hydrogel in the pipeline of the linear scaffold and the gap of the scaffold to simulate endoneurium. The bionic artificial nerve scaffold based on a bionic theory better simulates the structure of peripheral nerves and can promote and guide axon and Schwann cells to grow orderly and pass through damaged peripheral nerves based on a good mechanical property.
Description
Technical field
The invention belongs to the preparation field of tissue engineering artificial Nerve Scaffold, particularly a kind of preparation method of the biomimetic prosthetic Nerve Scaffold built by collagen protein.
Background technology
Along with the development of modern society, accident injury war earthquake tumor operation etc. causes the number of nerve injury to increase year by year, in the whole world every year newly-increased wound case, peripheral nerve injury case accounts for 1.5% ~ 4.0% China's peripheral nerve injury case newly-increased 60 ~ 900,000 examples every year, wherein need to repair neural case by neural transplantation and be about 300,000 ~ 450,000 examples, if malpractice causes life-long disabilities, serious commercial is caused to be born and the Reparation and Reconstruction of burden on society peripheral nerve defection is the development of a great problem along with tissue engineering of current wound surgery microsurgery, peripheral nerve tissue's engineering is also day by day carried out, being acknowledged as one of the effective means addressed 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, be used for guiding the nerve of neurologic defect near-end to extend regeneration, connect neurologic defect two ends, and the neurotrophic factor isoreactivity material of the infiltration and the secretion of allowable damage nervous tissue that reduce one-tenth fibrous tissue around diffuses into, and conduit is interior promotes neuranagenesis.
Oneself finds multiple neural substitution material up to now, comprises biomaterial as autologous tissue peripheral heart amniotic duct, allosome tissue's nervus vasculairs etc., achieve certain repairing effect but easily caused the immunological rejection of host due to these materials or easily subside, draw materials and purify and more complicated, and accurately can not control the length of conduit as required, diameter, the physical behaviors such as pipe thickness, limit its clinical practice current, what domestic and international research was more is Biodegradable material wherein collagen protein because of it, to have plasticity strong, immunological rejection is low, the advantages such as acquisition is convenient receive to be applied still more and more widely, the collagen-based materials of current report is due to the reason such as lack of alignment of pore size and collagen fiber, well can't guide the growth of regenerating nerve.
Summary of the invention
In order to solve above-mentioned Problems existing, the collagen-based materials that the present invention adopts 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 larger 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 larger application prospect in the clinical practice of neuranagenesis reparation
The object of the present invention is to provide a kind of preparation method of the biomimetic prosthetic Nerve Scaffold built by collagen protein.
The technical solution used in the present invention is:
A preparation method for the 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 in-25 ~-15 DEG C of freezing 60 ~ 80min after mould, then under vacuum-80 ~-60 DEG C of conditions lyophilizing 15 ~ 25min; Take out to obtain collagen-based materials;
Above-mentioned collagen-based materials is stretched to 1.5 ~ 2.5 times of raw footage, then in a stretched state, be placed on-25 ~-15 DEG C of freezing 60 ~ 80min, secondary freeze drying 1.5 ~ 2.5h is carried out under putting into vacuum-80 ~-60 DEG C of conditions again, obtain the inner line style support containing orderly trickle pipeline, namely orderly collagen scaffold, for subsequent use;
The preparation of nano-fiber film: the hexafluoroisopropanol solution of preparation collagen protein powder, makes nano-fiber film by this solution, for subsequent use;
The preparation of collagen hydrogel: prepare collagen hydrogel; For subsequent use;
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 use all gaps in collagen hydrogel filling tube with analog neuron inner membrance, biomimetic prosthetic Nerve Scaffold can be obtained.
Further, above-mentioned steps 1) described in collagen-based materials be the column collagen-based materials of 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, adopt 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, the collagenic aqueous solution of the method preparing collagen hydrogel to be compound concentration be 0.8 ~ 1.2mg/ml, can obtain collagen hydrogel.
Further, above-mentioned steps 2) in, nano-fiber film is rolled into the tubulose that diameter is 1 ~ 2mm, length is 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 prepared of the present invention based on bionics theory simulate perineural supporting structure, ensureing on good mechanical property basis, can promote and guide aixs cylinder and Scs to grow in an orderly manner and by the peripheral nerve of defect.
2) the 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 the growth guiding nerve tract simultaneously, incorporating collagen itself has the low feature of degradable immunological rejection, 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 the oriented growth guiding neural axon under the effect of pulling force; In periphery, with the larger collagen nanofiber thin-film material of hardness as support, can ensure 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 tool of collagen-based materials of the present invention in drawing process situation of change (A ~ C) originally;
Fig. 3 is the outer surface situation (A and B) of biomimetic prosthetic Nerve Scaffold and the visual condition (C) of orderly collagen scaffold of embodiment 1 preparation;
Fig. 4 is the scanning electron microscope (SEM) photograph of orderly collagen scaffold longitudinal section structure prepared by embodiment 1;
Fig. 5 is the cross section scanning electron microscope (SEM) photograph (A) that embodiment 1 prepares collagen scaffold prepared by the scanning electron microscope (SEM) photograph (B) of the inner Structure of cross section of biomimetic prosthetic Nerve Scaffold and 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 of repairing; B does the result of repairing with the biomimetic prosthetic Nerve Scaffold of this bright preparation, and C figure is the result of negative control group.
Detailed description of the invention
Below in conjunction with specific embodiment, further elaboration the present invention should understand, these embodiments are only not used in for illustration of the present invention to limit the scope of the invention should be understood in addition, after the content of having read the present invention's instruction, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's 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 in-20 DEG C of freezing 1h after mould, then put into freezer dryer vacuum-70 DEG C of lyophilizing 20min; Take out to obtain the column collagen-based materials of long 1cm diameter 1mm;
2) (be market existing equipment by above-mentioned collagen-based materials drawing device, the program control horizontal microelectrode of U.S.'s Sutter InstrumentP97 type can be selected to draw instrument) with each second 1.2mm speed slow stretching to the twice (stretching schematic diagram as shown in Figure 1) of raw footage, the concrete change procedure of collagen-based materials in drawing process is as shown in Fig. 2 A ~ C, then together with equipment at-20 DEG C of freezing 1h, be placed in vacuum-70 DEG C of secondary freeze drying 2h again, obtain the inner line style support (i.e. orderly collagen scaffold, as shown in Fig. 3 C and Fig. 4) containing orderly trickle pipeline; For subsequent use;
3) dissolved by collagen protein powder (commercial) hexafluoroisopropanol, in gained solution, collagen concentration is 100mg/ml, and this solution adopts electrostatic spinning technique to make the nano-fiber film that thickness is 2mm; For subsequent use;
4) prepare the collagenic aqueous solution that concentration is 1mg/ml, obtain collagen hydrogel; For subsequent use;
5) above-mentioned nano-fiber film is held diameter 1.5mm polyethylene mould and be rolled into that diameter is 1.5mm, length is the tubulose of 1.2cm, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (i.e. orderly collagen scaffold) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of filling above-mentioned preparation in pipe, fill full collagen hydrogel with analog neuron inner membrance with microsyringe in the pipeline of final online type support (i.e. orderly 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, therefrom can find out that tube-surface is coarse, has comparatively uniform space can ensure the diffusive transport of nutrient substance and metabolic waste.Line style support (the i.e. orderly collagen scaffold) outward appearance containing orderly trickle pipeline prepared in the present embodiment, as shown in institute Fig. 3 C, therefrom can find out its surface display linear structure to a certain degree.
The longitudinal section structure of the line style support (i.e. orderly collagen scaffold) of the orderly trickle pipeline prepared by the present embodiment carries out scanning electron microscope detection, testing result as shown in Figure 4, therefrom can find out that its internal structure forms linear halfpipe, long axis direction obviously hinder growth walk crosswise partition.
The inner Structure of cross section of the biomimetic prosthetic Nerve Scaffold prepared by the present embodiment carries out scanning electron microscope detection, testing result as shown in Figure 5 B, therefrom can find out that cross section is the hole differed in size, pore diameter is 20-80 μm, explanation 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, do not have stretched, its internal structure is laminated structure at random; Wherein the concrete operations of existing conventional method can refer to di Summa PG, Kingham PJ,
etal.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,
etal. 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,
etal.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, and nano-fiber film inside includes orderly collagen scaffold and collagen gel
embodiment 2
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 25mg/ml, pours in-15 DEG C of freezing 80min after mould, then puts into freezer dryer vacuum-60 DEG C of lyophilizing 25min; 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 each second 1.4mm speed slow stretching to 2.5 times of raw footage, then at extended state, be placed on-15 DEG C of freezing 80min, put into-80 DEG C, vacuum again and carry out secondary freeze drying 1.5h, obtain the inner line style support containing orderly trickle pipeline, namely orderly collagen scaffold, for subsequent use;
3) preparation of nano-fiber film: collagen protein powder hexafluoroisopropanol is dissolved, in gained solution, collagen concentration is 80mg/ml, and this solution adopts electrostatic spinning technique to make the nano-fiber film that thickness is 2.2mm; For subsequent use;
4) preparation of collagen hydrogel: preparation concentration is the collagenic aqueous solution of 0.8mg/ml, obtains collagen hydrogel; For subsequent use;
5) above-mentioned nano-fiber film being held diameter 2mm polyethylene mould, to be rolled into diameter be 2mm, length is 1cm tubulose, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (i.e. orderly collagen scaffold) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of filling above-mentioned preparation in pipe, finally fill full collagen hydrogel with analog neuron inner membrance with microsyringe in the pipeline of orderly collagen scaffold and in gap, 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 in-25 DEG C of freezing 60min after mould, then puts into freezer dryer vacuum-80 DEG C of lyophilizing 15min; 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 each second 1mm speed slow stretching to 1.5 times of raw footage, then at extended state, be placed on-25 DEG C of freezing 60min, put into-60 DEG C, vacuum again and carry out secondary freeze drying 2.5h, obtain the inner line style support containing orderly trickle pipeline, namely orderly collagen scaffold, for subsequent use;
3) preparation of nano-fiber film: collagen protein powder hexafluoroisopropanol is dissolved, in gained solution, collagen concentration is 120mg/ml, and this solution adopts electrostatic spinning technique to make the nano-fiber film that thickness is 1.8mm; For subsequent use;
4) preparation of collagen hydrogel: preparation concentration is the collagenic aqueous solution of 1.2mg/ml, obtains collagen hydrogel; For subsequent use;
5) above-mentioned nano-fiber film is held diameter 1mm polyethylene mould and be rolled into that length is 1.5cm, diameter is the tubulose of 1mm, with analog neuron adventitia, then under stereomicroscope, with microforceps, the line style support (i.e. orderly collagen scaffold) of orderly trickle pipeline is contained with analog neuron bundle film in the inside of filling above-mentioned preparation in pipe, finally fill full collagen hydrogel with analog neuron inner membrance with microsyringe in the pipeline of orderly collagen scaffold and in gap, biomimetic prosthetic Nerve Scaffold can be obtained.
Further effect detection is done to biomimetic prosthetic Nerve Scaffold prepared by above-described embodiment below.
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, 1cm defect is done to rat right sciatic nerves, experimental group this biomimetic prosthetic Nerve Scaffold bridge joint is repaired, the empty set pipe that negative control group adopts simple collagen to roll, positive controls adopts the goldstandard nerve autograft of nerve injury treatment.All repair materials are all by micro suture bridge joint defect.Post operation one month, gets graft stage casing and does rip cutting.Repairing effect is evaluated by the situation detecting neural axon regeneration.
Testing result as shown in Figure 7, therefrom can be found out, A figure does the result of repairing with nerve autograft in positive controls, and the effect that its aixs cylinder (in figure shown in shiny red fluorescence) regenerates is best, and aixs cylinder density is large and linear arrangement is better.B figure does the result of repairing with the biomimetic prosthetic Nerve Scaffold of this bright preparation, the situation also better effect close to positive controls that aixs cylinder (in figure shown in shiny red fluorescence) regenerates, and the situation of aixs cylinder linear growth is comparatively obvious.C figure is the result of negative control group, then obviously far short of what is expected, almost can't see shiny red fluorescence, almost do not have the regeneration of aixs cylinder in figure.The above results illustrates that biomimetic prosthetic Nerve Scaffold prepared by the inventive method can each Rotating fields of analog neuron preferably, promotes the regeneration of neural axon in nerve injury.
For those skilled in the art's easy understand; the foregoing is only the preferred embodiment of patent of the present invention; not in order to limit the present invention, any amendment done within all the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain all dropping on application claims.
Claims (8)
1. a preparation method for the biomimetic prosthetic Nerve Scaffold built by collagen protein, is characterized in that: comprise the following steps:
1) preparation of orderly collagen scaffold: preparation concentration is the collagen solution of 15 ~ 25mg/ml, pours in-25 ~-15 DEG C of freezing 60 ~ 80min after mould, then under vacuum-80 ~-60 DEG C of conditions lyophilizing 15 ~ 25min; Take out to obtain collagen-based materials;
Above-mentioned collagen-based materials is stretched to 1.5 ~ 2.5 times of raw footage, then in a stretched state, be placed on-25 ~-15 DEG C of freezing 60 ~ 80min, secondary freeze drying 1.5 ~ 2.5h is carried out under putting into vacuum-80 ~-60 DEG C of conditions again, obtain the inner line style support containing orderly trickle pipeline, namely orderly collagen scaffold, for subsequent use;
The preparation of nano-fiber film: the hexafluoroisopropanol solution of preparation collagen protein powder, makes nano-fiber film by this solution, for subsequent use;
The preparation of collagen hydrogel: prepare collagen hydrogel; For subsequent use;
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 use all gaps in collagen hydrogel filling tube with analog neuron inner membrance, biomimetic prosthetic Nerve Scaffold can be obtained.
2. preparation method according to claim 1, is characterized 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.
3. preparation method according to claim 1, is characterized in that: in step 1), 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.
4. preparation method according to claim 1, is characterized in that: the concentration of the hexafluoroisopropanol solution of the powder of collagen protein described in step 1) is 80 ~ 120mg/ml.
5. preparation method according to claim 1, is characterized in that: in step 1), adopts electrostatic spinning technique that the hexafluoroisopropanol solution of collagen protein powder is made the nano-fiber film that thickness is 1.8 ~ 2.2mm.
6. preparation method according to claim 1, is characterized in that: in step 1), and the collagenic aqueous solution of the method preparing collagen hydrogel to be compound concentration be 0.8 ~ 1.2mg/ml, can obtain collagen hydrogel.
7. preparation method according to claim 1, is characterized in that: in step 2) in, nano-fiber film is rolled into the tubulose that diameter is 1 ~ 2mm, length is 1 ~ 1.5cm.
8. preparation method according to claim 1, is characterized 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 |
| 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 |
| 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 |
| CN109172036A (en) * | 2018-06-22 | 2019-01-11 | 中山大学附属第医院 | A kind of multichannel peripheral nerve conduit and preparation method thereof |
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| 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 |
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