CN107224620A - Carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its breeding method - Google Patents
Carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its breeding method Download PDFInfo
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Abstract
The present invention provides a kind of carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its breeding method, wherein, Bionic-type myocardial tissue includes：The cardiac muscle cell's layer being arranged in order from top to bottom, carbon nano-tube film, and basalis.The carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its breeding method of the present invention, cardiac muscle cell's cultivation timbering material is used as a result of the CNT of height-oriented property so that cardiac muscle cell forms the orientation growth of similar cardiac muscular tissue on the carbon nanotubes.Therefore Bionic-type myocardial tissue provided by the present invention can fully imitate the microstructure of cardiac muscle cell's orientation.Further, because CNT has good electric conductivity, effective connection path is provided therefore, it is possible to the transmission for iuntercellular electric signal, so that the Bionic-type myocardial tissue of the present invention has the cardiac muscular tissue for substituting conventional polymer Breeding, the application potential of the bionic heart sticking patch with cardiac synchronous pacing function is built.
Description
Technical field
The present invention relates to a kind of carbon nano-tube coextruded film electrode, the invention further relates to a kind of Bionic-type myocardial tissue, sheet
Invention further relates to the breeding method of Bionic-type myocardial tissue, belongs to field of tissue engineering technology.
Background technology
Myocardial infarction is called miocardial infarction, is due to coronary occlusion, and blood flow is interrupted, and the flesh that causes to divert one's attention is because serious is held
Continue property ischemic and occur local necrosis.The cardiac muscle of necrosis is gradually substituted by fibrous scar tissue, is triggered remodeling ventricle, is ultimately resulted in
Heart failure is even died suddenly.At present the treatment method that is used of clinic include drug therapy, it is PCI, thromboembolism treatment, coronal
Arterial bypass, although can delay remodeling ventricle, improves heart function, can not fundamentally repair damaged myocardium, blocking
Course advancement.Heart transplant is then because donor source is limited and immune rejection, it is impossible to meet clinical demand.It is above-mentioned to solve
Problem, researcher proposes organizational project cardiac patch concept.I.e. in laboratory culture multifunctional heart tissue, occur in patient
Damaged tissues are replaced before heart failure, so as to repair the 26S Proteasome Structure and Function of heart.Cardiac muscle tissue engineering research includes 3 sides
Face：Seed cell, timbering material and tissue construction and transplanting, many technological break-throughs have been obtained by studying for many years.It is all kinds of natural
And chemical synthesis high polymer material shows preferable biocompatibility, and there is a certain degree of adjustment effect to cellular activity.
However, multifunctional heart sticking patch really moves towards clinical practice from laboratory, also problems are urgently to be resolved hurrily.Particularly myocardium group
Knit with unique electrophysiological characteristics, and cardiac muscle cell heart it is shallow, in, deep three layers in different directions be orientated extension, electricity
Signal quickly and orderly conduction wherein causes cardiac synchronous to shrink.Traditional myocardium sticking patch hybridization scheme is difficult to fully simulation cardiac muscle
Orientation structure and electric signal conduction function.
The content of the invention
It is an object of the invention to provide a kind of carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its cultivation
Method, to solve the above problems.
Present invention employs following technical scheme：
The present invention provides a kind of carbon nano-tube coextruded film electrode, it is characterised in that including：CNT bonded to each other
CNT in film and basalis, the carbon nano-tube film has high orientation.
Further, carbon nano-tube coextruded film electrode of the invention, can also have the feature that：Wherein, the base
Bottom is flexible non-conductive macromolecule membrane, and material is silicon rubber, polytetrafluoroethylene (PTFE), wherein the one of polyethylene and polyvinyl chloride
Kind.
Further, carbon nano-tube coextruded film electrode of the invention, can also have the feature that：Wherein, the carbon
In nano-tube film, include 3~10 layers of single-layer carbon nano-tube.
Further, carbon nano-tube coextruded film electrode of the invention, can also have the feature that：Wherein, the carbon
The scope of the thickness sum of nano-tube film and the basalis is >=50 μm ,≤200 μm.
The present invention also provides a kind of Bionic-type myocardial tissue, including the CNT THIN COMPOSITE as described in above-mentioned any one
Membrane electrode, it is characterised in that including：The cardiac muscle cell's layer being arranged in order from top to bottom, carbon nano-tube film, and basalis,
Wherein, the direction of growth of the cardiac muscle cell in cardiac muscle cell's layer is consistent with the direction of orientation carbon nanotube film.
Further, Bionic-type myocardial tissue of the invention, can also have the feature that：Wherein, the basalis makes
The non-conductive macromolecule membrane of flexibility is replaced with sheet glass.
The present invention also provides a kind of breeding method of Bionic-type myocardial tissue, it is characterised in that comprise the following steps：
Step one, height-oriented carbon nano-tube film is prepared, the substrate of extracorporeal culture cardiac muscle cell is used as；
Step 2, carbon nano-tube film is attached on sheet glass or non-conductive macromolecule membrane；
Cardiac muscle cell, separating myocardium cell, is inoculated on carbon nano-tube film by step 3.
Further, the breeding method of Bionic-type myocardial tissue of the invention, can also have the feature that, in addition to：
Step 4: cells into cardiomyocytes implements the electric pulse stimulation of simulation heart.
Further, the breeding method of Bionic-type myocardial tissue of the invention, can also have the feature that：Wherein, it is electric
The voltage of impulse stimulation is 2~6V/cm, and pulse width is 2ms；Pulse frequency is 60~150bmp.
Further, the breeding method of Bionic-type myocardial tissue of the invention, can also have the feature that：Wherein, it is electric
Impulse stimulation is applied by carbon nano-tube film cells into cardiomyocytes.
The beneficial effect of invention
The carbon nano-tube coextruded film electrode and Bionic-type myocardial tissue and its breeding method of the present invention, as a result of height
The CNT of orientation is spent as cardiac muscle cell's cultivation timbering material so that cardiac muscle cell forms similar on the carbon nanotubes
The orientation growth of cardiac muscular tissue.Therefore Bionic-type myocardial tissue provided by the present invention can fully imitate cardiac muscle cell's orientation
Microstructure.Further, because CNT has good electric conductivity, carried therefore, it is possible to the transmission for iuntercellular electric signal
For effective connection path so that Bionic-type myocardial tissue of the invention, which has, substitutes conventional polymer cell culture support material
Material, builds the application potential of multifunctional bionic cardiac patch.
Brief description of the drawings
Fig. 1 is the structural representation of Bionic-type myocardial tissue；
Fig. 2 is the electron microscope for the aligned carbon nanotube prepared；
Fig. 3 a are the fluorescent microscopy images that aligned carbon nanotube guides cardiac muscle cell's oriented growth, when cultivating the 3rd day
Fluorescent microscopy images；
Fluorescent microscopy images when Fig. 3 b is cultivate the 7th day；
When Fig. 3 c is cultivate the 7th day, the photo of white light is opened under fluorescence microscope simultaneously, it is seen that cytochrome oxidase isozymes direction and carbon
Nanotube orientation direction is consistent.
Fig. 4 a are the three cluster cells depended on orientation carbon nanotube film observed under microscope, scale：500μm；
Fig. 4 b are the spontaneous beat curve of this three clusters cell；
Fig. 4 c are applied by carbon nano-tube film to these cells after pacing signal, this corresponding pacing frequency of three clusters cell
The beating change curve of (60bpm and 100bpm)；
Fig. 5 is the photo that flexible electrode is closely attached to heart；
Fig. 6 is the photo by three hearts of flexible electrode simultaneous pacing.
Embodiment
Illustrate the embodiment of the present invention below in conjunction with accompanying drawing.
1. carbon nano-tube coextruded film electrode
The preparation method of 1.1 carbon nano-tube films：
Height-oriented carbon nano-tube film is prepared according to prior art method, i.e., carbon nanometer is prepared by chemical vapour deposition technique
Pipe array, then corresponding fiber and film are prepared into by dry spinning.Preparation method is：First in a quartz tube furnace, with
It is coated with Fe (1.2nm)/Al2O3The silicon chip of (3nm) is as catalyst, using ethylene gas as carbon source, with high-purity argon gas and High Purity Hydrogen
Gas gaseous mixture is warming up to 740 degrees Celsius, CNT starts growth as carrier gas.Growth time was controlled at 10-20 minutes, cold
But to room temperature.Obtain spinning after carbon nano pipe array, pull out individual layer from the edge of carbon nano pipe array with a blade and battle array
Row are with wide carbon nano-tube film.The carbon nano-tube film pulled straight out shows that observation has significant under height-oriented property, microscope
Orientation structure.Make to prepare such carbon nano-tube film in this way, referred to as super-aligned carbon
Nanotube sheet, i.e., height-oriented property CNT.The height-oriented property CNT prepared is as shown in Figure 2.This hair
Bright research finds, the carbon nano-tube film with height-oriented property prepared by dry spinning technology, with extremely light quality, good
Modulatory character in good pliability and self-supporting, porous coarse surface, excellent electric conductivity and nanoscale.
1.2 carbon nano-tube films are compounded to form carbon nano-tube coextruded film electrode with substrate
The orientation carbon nanotube film that dry spinning is pulled straight out is placed directly within substrate.
Substrate is divided into non-flexible substrates and flexible substrates.Non-flexible substrates can use the cover glass or glass of cell culture
Piece.Flexible substrates use non-conductive macromolecule membrane, including silicon rubber, polytetrafluoroethylene (PTFE), polyethylene, polyvinyl chloride etc..
Substrate thickness is optimal less than 200 μm between 10-1000 μm；Size of foundation base is according to culture dish size, or needs
The mammalian heart size control fought, in 0.2*0.2cm2And 2*2cm2Between can the random change of Non-scale constraint.
Because electrostatic attraction and Van der Waals power are acted on, orientation carbon nanotube film has the attached property of good note to above-mentioned substrate
Matter.Also a small amount of absolute ethyl alcohol can be added dropwise, volatilizes naturally and plays more preferable fixation.Carbon nano-tube film in substrate can multilayer
Superposition.The general superposition number of plies is between 3-10 layers.The film of individual layer is equivalent to the nanometer grid that single-root carbon nano-tube is constituted, and film is thick
Degree is only 10 rans.Many gaps are had between CNT.And relatively fragile, easy fragmentation when bending.
Multilayer can be effectively improved this problem.Importantly, single thin film resistance is larger, film is improved by multi-laminate Calais
Electrical conductivity.Also controllability is improved simultaneously.
Orientation carbon nanotube film in flexible substrates can 90 degree of bendings without coming off or crushing.First aligned carbon nanotube
To bending 500 times in side, Thin film conductive rate hardly changes.During extroversion bending, bending for the first time can be caused
20% electrical conductivity reduction, continues thereafter with extroversion and is folded into 500 times, electrical conductivity is no longer produced and significantly reduced.Inflexion and outer
It is as follows to the difference of bending：Carbon nano-tube film note is attached in the substrate of flexibility, towards the bending of this face of carbon nano-tube film, cries interior
To bending, towards the bending of that face of substrate, export-oriented bend is cried.Vivider, when CNT is upper, flexible substrates are under, and U-shaped is just
It is inflexion, n shapes are exactly export-oriented bending.
Orientation carbon nanotube film note is attached on flexible non-conductive macromolecule and carbon nano-tube coextruded film is made, and can be applied to
The electrode of pacemaker.Carbon nano-tube coextruded film is cut into according to the organ size of required pace-making by box-like size.Carbon is received
Mitron laminated film shows good flexibility, can multi-angle bending and significant change does not occur for structure.CNT THIN COMPOSITE
Film thickness is controlled between 10-1000 μm, and carbon nanotube layer thickness is less than 1 μm, can be neglected.The non-conductive high score of major regulatory
Molecular layers thick.Macromolecule membrane can directly buy the silicon rubber of the commodity production of thickness customization, polytetrafluoroethylene (PTFE), polyethylene, poly-
The films such as vinyl chloride.Also can purchase commercial formulation precursor solution and initiator, laboratory by product description ratio oneself
The smooth container film forming in bottom is poured into after row mixing.Carbon nano-tube coextruded film thickness is less than 200 μm most preferably, now can closely paste
Close soft histoorgan surface.But film thickness is too thin will to increase transfer operation difficulty, so thickness is generally higher than 50 μm.
Prepared aligned carbon nanotube composite film electrode, can the multiple isolated hearts of synchronous pacing.One typical example
As shown in Figure 6:3 suckling mouse hearts of 1-3 days are removed and pass through tyrode lavation, are placed on non-conductive platform.Make to take
Three hearts are covered simultaneously to carbon nano-tube compound film flexible electrode, show good stickiness.Connect pacemaker, three hearts
It is dirty in 2ms, 5-10V can synchronous pacing under the conditions of 1-3Hz.Pace-making situation can in real time be supervised by ventricular muscles individual event action potential
Survey.On the other hand, open chest surgery under microscope is carried out to test mice under anesthesia, flexible electrode is directly attached to the heart
Dirty surface, combination electrode also can successfully pace heart.Above-mentioned experimental result shows, flexible orientation carbon nanometer prepared by the present invention
Pipe compound film electrode, the potentiality in cardiac synchronous pace-making with further application and development.
2. Bionic-type myocardial tissue
Structure is as shown in figure 1, Bionic-type myocardial tissue includes：Cardiac muscle cell's layer 11 and carbon nano-tube coextruded film electrode
12.Cardiac muscle cell's layer 11 is covered on carbon nano-tube coextruded film electrode 12.
3. the breeding method of Bionic-type myocardial tissue
The cardiac muscle of mammal cell of separation is obtained according to this area conventional method, or passes through commercially available approach and preservation mechanism
Obtain.By taking typical SD neonatal rat cardiomyocytes exposeds cultivating system as an example.Cardiac muscle cell is dispersed in corresponding regular growth culture
In liquid, it is inoculated in bottom and is covered with the Tissue Culture Dish of orientation carbon nanotube film or blake bottle.Using thin in typical method
Born of the same parents cultivate six orifice plates, and a piece of 1.5*1.5cm for being covered with orientation carbon nanotube film is placed in each bottom hole portion2Cover glass.Cell kind
Plant amount is 1 × 106Individual cells/well.The plantation concentration of cell can be adjusted, and culture dish is placed on conventional such as 37 DEG C/5% dioxy
Change in carbon incubator.Cardiac muscle cell independently settles note and is attached to orientation carbon nanotube film surface, automatic sensing aligned carbon nanotube
Film surface topological structure simultaneously depends on the growth of its orientation.Cell the 3rd day is to show significantly to take along CNT cultivating
The shape characteristic extended to direction, as shown in Fig. 3 a, Fig. 3 b and Fig. 3 c.Cell growth status passes through fluorescence after immunofluorescence dyeing
MIcrosope image is observed.It can be seen that cardiac muscle cell shows the form of orientation growth.
Orientation carbon nanotube film has significant guiding function to the growth of cardiac muscle cell's orientation.Cell culture the 7th day,
Cell volume increases, and obvious muscle segment band occurs, illustrates that eucaryotic cell structure tends to be ripe.But the orientation pattern of cell is still
It is good to keep.Obvious Cell tracking PROTEIN C X43 expression can be detected by routine immunization fluorescent staining.CX43 is to adjacent
The transmission of intercellular signal plays an important roll.The CX43 of cardiac muscle cell's expression of orientation growth is also distributed in side wallization, edge
The arrangement mode that cell edges show orientation.
Aligned carbon nanotube specifically has good electric conductivity.In the present invention in cell cultivation process, i.e., cell attachment is complete
Entirely, the 3rd day when, by by orientation carbon nanotube film substrate connect external signal transmitter, to cell carry out it is lasting
Electric-impulse stimulation.Electro photoluminescence scope：Pace-making vector 2-6V/cm；Pulsewidth, 2ms；Frequency, 60-150bpm.In order to prevent electric leakage, outside
The coupling part of circuit and orientation carbon nanotube film uses non-conductive macromolecule, and such as silicon rubber is protected.Ensure electric signal
Pass through the cardiac muscle cell in the incoming cultivation of aligned carbon nanotube substrate.By thermal imaging system it was observed that, cell culture system is held
Continuous energization does not result in extra fever phenomenon.Compared with without electro photoluminescence, electro photoluminescence culture does not bring obvious Apoptosis yet
The increase of quantity.On the other hand, the culture of electro photoluminescence auxiliary improves CX43 expression quantity (than not stimulating high 30-50%).Electricity thorn
Swash and the pattern of cell on aligned carbon nanotube is had no significant effect.
Regardless of whether being powered, the cardiac muscle cell cultivated by aligned carbon nanotube can show that obvious synchronization certainly
Hair beating phenomenon, SD neonatal rat cardiomyocytes exposeds are about 80~200bpm cultivating the 3rd day synchronous spontaneous beat frequency, are such as schemed
Shown in 3a, b.3-4 days after incubation, electrophysiology in situ is carried out for the cardiac muscle cell of culture by patch clamp technique and commented
Estimate, show that orientation carbon nanotube film can promote the maturation of electrophysiological characteristics of neonatal rat myocardial cell.Electrophysiological characteristics
Ripe standard is：Resting potential -70~-80mV, 90~110mV of action potential amplitude, the time-histories of action potential multipole 90%
(APD90) 130~160ms)；Simultaneously orientation carbon nanotube film can also reduce between different cardiac muscle cells, same cardiac muscle cell
Electrophysiological heterogeneity (APD between continuous several times beating30And APD90It is heterogeneous compared with cover glass reduction by 60~80%).By taking
To carbon nano-tube film substrate, pace-making can be synchronized to all cells, as illustrated in fig. 4 c.Fig. 4 a be microscope under observe
The three cluster cells depended on orientation carbon nanotube film, scale：500μm；Fig. 4 b are the spontaneous beat curve of this three clusters cell；
Fig. 4 c are applied by carbon nano-tube film to these cells after pacing signal, this corresponding pacing frequency of three clusters cell (60bpm and
Beating change curve 100bpm).
As shown in figure 5, the Bionic-type myocardial tissue provided according to the method for the present invention, can be good at being attached to cardiac muscle
On, realize the effect paced in vivo.
In summary, present invention firstly provides can guide cardiac muscle cell's oriented growth, mould using carbon nano-tube compound film
Intend the connexin expression and side wall between the microcosmic myocardial structural of naive hearts, increase cardiac muscle cell, and between cardiac muscle cell
The quick conduction of electric signal provides physical channel.Meanwhile, carbon nano-tube compound film promotes neonatal rat myocardial cell electrophysiological characteristics
Maturation, reduces cardiac muscle cell's repolarization dispersion, and all cardiac muscle cells contributed on carbon nano-tube compound film produce spontaneous, rule
Beating, it is to avoid the generation of arrhythmia cordis.Electro photoluminescence collaboration carbon nano-tube compound film can promote the maturation of neonatal rat myocardial cell.
Inside and outside pace-making experiment also shows that the one chip carbon nano-tube compound film electrode frivolous, elasticity is splendid also has excellent tissue
Attachment and pacing function.Therefore, carbon nano-tube compound film cardiac repair after heart infarction is treated and with the heart infarction of block
Had a wide range of applications afterwards in the following cardiac resynchronization therapy of heart failure.
Claims (10)
1. a kind of carbon nano-tube coextruded film electrode, it is characterised in that including：
CNT in carbon nano-tube film and basalis bonded to each other, the carbon nano-tube film has high orientation.
2. carbon nano-tube coextruded film electrode as claimed in claim 1, it is characterised in that：
Wherein, the basalis is flexible non-conductive macromolecule membrane, and material is silicon rubber, polytetrafluoroethylene (PTFE), polyethylene and
The one of which of polyvinyl chloride.
3. carbon nano-tube coextruded film electrode as claimed in claim 1, it is characterised in that：
Wherein, in the carbon nano-tube film, including 3~10 layers of CNT.
4. carbon nano-tube coextruded film electrode as claimed in claim 1, it is characterised in that：
Wherein, the scope of the thickness sum of the carbon nano-tube film and the basalis is >=50 μm ,≤200 μm.
5. a kind of Bionic-type myocardial tissue, including the carbon nano-tube coextruded film electricity as described in any one in claim 1-4
Pole, it is characterised in that including：
It is arranged in order from top to bottom
Cardiac muscle cell's layer,
Carbon nano-tube film,
And basalis,
Wherein, the direction of growth of the cardiac muscle cell in cardiac muscle cell's layer is consistent with the direction of orientation carbon nanotube film.
6. Bionic-type myocardial tissue as claimed in claim 5, it is characterised in that：
Wherein, the basalis replaces flexible non-conductive macromolecule membrane using sheet glass.
7. a kind of breeding method of Bionic-type myocardial tissue, it is characterised in that comprise the following steps：
Step one, height-oriented carbon nano-tube film is prepared, the substrate of extracorporeal culture cardiac muscle cell is used as；
Step 2, carbon nano-tube film is attached on sheet glass or non-conductive macromolecule membrane；
Cardiac muscle cell, separating myocardium cell, is inoculated on carbon nano-tube film by step 3.
8. the breeding method of Bionic-type myocardial tissue as claimed in claim 7, it is characterised in that also include：
Step 4: cells into cardiomyocytes implements the electric pulse stimulation of simulation heart.
9. the breeding method of Bionic-type myocardial tissue as claimed in claim 8, it is characterised in that：
Wherein, the voltage of electric pulse stimulation is 2~6V/cm, and pulse width is 2ms；Pulse frequency is 60~150bmp.
10. the breeding method of Bionic-type myocardial tissue as claimed in claim 9, it is characterised in that：
Wherein, electric pulse stimulation is applied by carbon nano-tube film cells into cardiomyocytes.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110201233A (en) * | 2019-06-17 | 2019-09-06 | 嘉兴莱普晟医疗科技有限公司 | A kind of method and its application preparing myocardium sticking patch |
| CN110507903A (en) * | 2019-09-10 | 2019-11-29 | 上海交通大学医学院附属新华医院 | Multifunctional membrane based on carbon nanotube and preparation method thereof and power assist apparatus |
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| CN115216438A (en) * | 2022-06-28 | 2022-10-21 | 苏州大学 | Preparation method and application of engineering myocardial tissue based on big blue flashing butterfly wing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110201233A (en) * | 2019-06-17 | 2019-09-06 | 嘉兴莱普晟医疗科技有限公司 | A kind of method and its application preparing myocardium sticking patch |
| CN110201233B (en) * | 2019-06-17 | 2021-06-25 | 嘉兴莱普晟医疗科技有限公司 | Method for preparing myocardial patch and application thereof |
| CN110507903A (en) * | 2019-09-10 | 2019-11-29 | 上海交通大学医学院附属新华医院 | Multifunctional membrane based on carbon nanotube and preparation method thereof and power assist apparatus |
| CN110507903B (en) * | 2019-09-10 | 2023-05-30 | 上海交通大学医学院附属新华医院 | Multifunctional membrane based on carbon nano tube, preparation method thereof and power auxiliary device |
| CN112588212A (en) * | 2020-11-20 | 2021-04-02 | 南京鼓楼医院 | Preparation method of oriented carbon nanotube hydrogel film for myocardial cell culture |
| CN112657051A (en) * | 2020-12-25 | 2021-04-16 | 上海交通大学医学院附属上海儿童医学中心 | Biological type ventricular assist pump based on conjugate coaxial electrostatic spinning technology and preparation method thereof |
| CN112657051B (en) * | 2020-12-25 | 2023-08-04 | 上海交通大学医学院附属上海儿童医学中心 | Biological type ventricular assist pump based on conjugate coaxial electrostatic spinning technology and preparation method thereof |
| CN115216438A (en) * | 2022-06-28 | 2022-10-21 | 苏州大学 | Preparation method and application of engineering myocardial tissue based on big blue flashing butterfly wing |
| CN115216438B (en) * | 2022-06-28 | 2024-05-10 | 苏州大学 | Preparation method and application of engineered myocardial tissue based on bluish-flash wings |
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