CN108642576A - A kind of electrospinning process and electrospun fibers of extracellular matrix - Google Patents
A kind of electrospinning process and electrospun fibers of extracellular matrix Download PDFInfo
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- CN108642576A CN108642576A CN201810467964.5A CN201810467964A CN108642576A CN 108642576 A CN108642576 A CN 108642576A CN 201810467964 A CN201810467964 A CN 201810467964A CN 108642576 A CN108642576 A CN 108642576A
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- 210000002744 extracellular matrix Anatomy 0.000 title claims abstract description 99
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 title claims abstract description 97
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 title claims abstract description 97
- 239000000835 fiber Substances 0.000 title claims abstract description 74
- 238000000034 method Methods 0.000 title claims abstract description 42
- 230000008569 process Effects 0.000 title claims abstract description 34
- 238000001523 electrospinning Methods 0.000 title claims abstract description 23
- 238000010041 electrostatic spinning Methods 0.000 claims abstract description 56
- 239000000843 powder Substances 0.000 claims abstract description 50
- 210000004027 cell Anatomy 0.000 claims abstract description 48
- 239000011259 mixed solution Substances 0.000 claims abstract description 48
- 238000009987 spinning Methods 0.000 claims abstract description 46
- 239000000243 solution Substances 0.000 claims abstract description 44
- 239000000463 material Substances 0.000 claims abstract description 35
- 239000002798 polar solvent Substances 0.000 claims abstract description 17
- 229910052731 fluorine Inorganic materials 0.000 claims abstract description 13
- 239000011737 fluorine Substances 0.000 claims abstract description 13
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000011017 operating method Methods 0.000 claims abstract description 4
- 238000000498 ball milling Methods 0.000 claims description 23
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 12
- 238000004132 cross linking Methods 0.000 claims description 12
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 claims description 6
- 210000000578 peripheral nerve Anatomy 0.000 claims description 6
- 230000003068 static effect Effects 0.000 claims description 6
- 230000000968 intestinal effect Effects 0.000 claims description 4
- 210000004876 tela submucosa Anatomy 0.000 claims description 4
- 238000010009 beating Methods 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 abstract description 11
- 238000009826 distribution Methods 0.000 abstract description 10
- 238000007306 functionalization reaction Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 description 14
- 239000000725 suspension Substances 0.000 description 14
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- 238000002156 mixing Methods 0.000 description 8
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- 239000011324 bead Substances 0.000 description 2
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- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 1
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- 239000004744 fabric Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
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- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
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Classifications
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
Landscapes
- Engineering & Computer Science (AREA)
- Mechanical Engineering (AREA)
- Textile Engineering (AREA)
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Spinning Methods And Devices For Manufacturing Artificial Fibers (AREA)
- Artificial Filaments (AREA)
Abstract
The present invention relates to electrostatic spinning technique fields, provide a kind of electrospinning process of extracellular matrix, and operating procedure includes：The extracellular matrix powder of cell will be gone by 0.03~0.04g:1mL is mixed with fluorine-containing highly polar solvent, obtains mixed solution；Mixed solution is not added to high molecular material and carries out the obtained electrospun fibers of electrostatic spinning as spinning solution.Fiber made from this method has the performance of micro-nano multistage fiber, and since spinning solution does not add high molecular material therefore is conducive to cell attachment, differentiation and functionalization.The present invention also provides one kind by electrospun fibers made from the above method, which has the performance of micro-nano multistage fiber, and distribution of fiber diameters is wider, and the natural degree of fiber is high, the characteristics of being more advantageous to cell attachment, differentiation and functionalization.
Description
Technical field
The present invention relates to electrostatic spinning technique fields, in particular to a kind of electrospinning process of extracellular matrix
And electrospun fibers.
Background technology
Organizational project (tissue engineering) is the multi-crossed disciplines based on cell biology and material science
Emerging science, carry out the research for building tissue or organ in vitro or in vivo.Organizational project is that tissue damage reparation proposes newly
Thinking.And the core in organizational project is to build the tissue engineering bracket of good biocompatibility, is cell transplantation, injury repair
Good carrier is provided.
The natural tissues extracellular matrix of human body is with nanofibrous structures, and nanofibrous structures are conducive to the attached of cell
It, and physical signal guiding cell function can be provided for cell.Based on bionical consideration, it is necessary to moulds for tissue engineering bracket
The nanofibrous structures of imitative extracellular matrix.And electrostatic spinning is a kind of processing prepares the mature technology of nano-fiber material, it is quiet
Electrospun this have the advantage that：Easy to operate, it is extensive to have industrialization for form, the structure snd size of controllable nanofiber
The potential of production, is therefore widely used in and prepares tissue engineering bracket.
The material that can be used for electrostatic spinning processing is broadly divided into synthetic material and natural material, including protein, polysaccharide, core
Acid and biological active matter.The machinability of synthetic material is got well than natural material.The spinning solution of electrostatic spinning is usually liquid
Or uniform solution or lotion.The property and ingredient of spinning solution are to electrostatic spinning process and finally obtained fiber morphology and life
Change performance and play very crucial influence, it usually needs there is suitable surface tension and viscosity, therefore also limit quiet
The material selection range of Electrospun.
When preparing organizational project nano fiber scaffold by electrostatic spinning, other than considering that physical arrangement is bionical, it is also necessary to
Consider bionical on ingredient.Extracellular matrix be in natural tissues by cell be secreted into it is extracellular, be present between cell big point
Son, complicated component, main component include that structural proteins (collagen, elastin laminin), single-minded albumen (fibrin etc.) and albumen are poly-
Sugar, and include a variety of growth factors.It is important that these ingredients maintain normal morphology, proliferation, differentiation and function words to play in cell
Adjustment effect.
Extracellular matrix be a kind of good material of bioactivity (obtained after human or animal tissues are directly taken off cell, because
This remains many natural active constituents), but current technology can not will go cellular matrix effect main material progress
Electrostatic spinning is processed.
In consideration of it, special propose the application.
Invention content
The present invention provides a kind of electrospinning process of extracellular matrix, it is intended to improve existing electrostatic spinning technique not
Can be to go cellular matrix to carry out electrostatic spinning as main material the problem of.
The present invention also provides a kind of electrospun fibers, the performance with micro-nano multistage fiber, fibre diameter point
Cloth is wider, and the natural degree of fiber is high, the characteristics of being more advantageous to cell attachment, differentiation and functionalization.
The invention is realized in this way：
A kind of electrospinning process of extracellular matrix, operating procedure include：
The extracellular matrix powder of cell will be gone by 0.03~0.04g:1mL is mixed with fluorine-containing highly polar solvent, is mixed
Solution；Mixed solution is not added to high molecular material and carries out the obtained electrospun fibers of electrostatic spinning as spinning solution.
Further, in preferred embodiments of the present invention, mixed solution is placed in ball mill, it is Celsius -12~-8
It does not add high molecular material under conditions of degree, 50~80HZ after 10~60min of ball milling and carries out electrostatic spinning as spinning solution.
Further, in preferred embodiments of the present invention, after mixed solution is stirred 2~7 days, it is placed in ball mill
Middle carry out ball-milling treatment.
Further, in preferred embodiments of the present invention, highly polar solvent includes trifluoroethanol or hexafluoroisopropanol.
Further, in preferred embodiments of the present invention, the operating condition for carrying out electrostatic spinning is：Syringe injection speed
Degree is 1mL/h~4mL/h, and distance is 6~10cm between syringe needle and reception tablet, and syringe needle adds 6~12kV of positive voltage, receiver board to apply
Add 0.5~1.5kV of negative voltage.
Further, in preferred embodiments of the present invention, electrostatic spinning operating process uses No. 9 flat mouth syringe needles of national standard.
Further, further include by electrostatic spinning obtained in preferred embodiments of the present invention, after electrostatic spinning
Fiber uses 2~12h of glutaraldehyde vapor crosslinking.
Further, in preferred embodiments of the present invention, the extracellular matrix powder that will remove cell with it is fluorine-containing highly polar
Further include before solvent mixing：The extracellular matrix of cell will be gone to carry out beating powder, 30~50 mesh are crossed and sieve to obtain the cell of cell
Epimatrix powder.
Further, in preferred embodiments of the present invention, extracellular matrix is that pig source property small intestinal submucosa is extracellular
Matrix or pig source property peripheral nerve extracellular matrix.
A kind of electrospun fibers are made using the electrospinning process of above-mentioned extracellular matrix.
The beneficial effects of the invention are as follows：The electrospinning process for the extracellular matrix that the present invention is obtained by above-mentioned design,
In use, due to being mixed first with appropriate proportioning with fluorine-containing polar solvent in the process, then in the feelings for not adding high molecular material
Electrostatic spinning is carried out as spinning solution using extracellular matrix as main material under condition, and makes final electrospun fibers obtained
Performance with micro-nano multistage fiber, distribution of fiber diameters is wider, and due to the natural degree of the spinning solution of the invention used
The bioactive ingredients of height, reservation are more, so it is more advantageous to the effect of cell attachment, differentiation and functionalization.
The electrospun fibers that the present invention is obtained by above-mentioned design, due to being made using method provided by the invention, because
This its performance with micro-nano multistage fiber, distribution of fiber diameters is wider, and is more advantageous to cell attachment, differentiation and work(
The characteristics of capable of changing.
Description of the drawings
It, below will be to required use in embodiment in order to illustrate more clearly of the technical solution of embodiment of the present invention
Attached drawing be briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not to be seen as
It is the restriction to range, it for those of ordinary skill in the art, without creative efforts, can be with root
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the scanning electron microscope (SEM) photograph for the electrospun fibers that the embodiment of the present invention 1 provides；
Fig. 2 is the scanning electron microscope (SEM) photograph for the electrospun fibers that the embodiment of the present invention 2 provides.
Specific implementation mode
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
A kind of electrospinning process of extracellular matrix is provided to the embodiment of the present invention below to be specifically described.
A kind of electrospinning process of extracellular matrix, operating procedure include：
S1, the extracellular matrix powder of cell will be gone by 0.03~0.04g:1mL is mixed with fluorine-containing highly polar solvent, is mixed
Close solution.
It goes extracellular matrix to derive from natural tissues, remains most of ingredient in natural tissues, being one kind has very
Good bioactivity and life compatibility and can be with the material of degradation in vivo.
Specifically, the extracellular matrix for having already passed through Cell extraction is carried out playing powder processing, beats powder and ball mill can be used
Low temperature beats powder or pulverizer beats powder.30~50 mesh sieve is crossed after beating powder, obtains the more uniform extracellular matrix powder of grain size, grain size
Excessive powder, which can return to, to be continued to beat powder.Preferably, extracellular matrix can be selected small intestinal submucosa extracellular matrix,
Peripheral nerve-cell epimatrix, it is highly preferred that pig source property small intestinal submucosa extracellular matrix or pig can be selected in extracellular matrix
Source property peripheral nerve extracellular matrix.It should be noted that in the present invention, other extracellular matrixs are same as primary raw material
Sample has same effect.
Extracellular matrix powder is weighed after sieving, and is added into fluorine-containing highly polar solvent, and the two is made to carry out
It is mixed to get mixed solution.The proportioning of extracellular matrix powder and fluorine-containing highly polar solvent is 0.03~0.04g:1mL.Preferably,
Fluorine-containing highly polar solvent includes trifluoroethanol or hexafluoroisopropanol.That is, fluorine-containing highly polar solvent may include trifluoroethanol or six
Fluorine isopropanol or the two mixture.
Due to going cellular stromal component complicated, there are more insoluble components.For make in mixed solution extracellular matrix with
Solvent is more fully mixed, and the soluble powder in extracellular matrix is made to be substantially soluble in solution, it is insoluble be divided into be uniformly suspended in
In solution.Mixed solution is stirred after the two mixing, is stirred 2~7 days.
Mixed solution after stirring is placed in the centrifuge tube of 2mL, steel ball or zirconia ball is added, then will
Centrifuge tube is put into progress low temperature ball milling homogenate in ball mill.The condition of ball milling is：Ball milling temperature is -12~-8 degrees Celsius, equipment
Frequency is 50~80HZ, and Ball-milling Time is 10~60min.It can ensure under above-mentioned ball milling condition in thorough break up cell epimatrix
Insoluble part in highly polar solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
S2, mixed solution is not added to high molecular material as the obtained electrospun fibers of spinning solution progress electrostatic spinning.
Specifically, mixed solution made from S1 is not added into high molecular material and is made quiet as spinning solution progress electrostatic spinning
Electricity spinning fibre.Existing extracellular matrix spinning solution is added with spinning solution made from high molecular material.The present invention provides
Extracellular matrix and electrospinning process in the spinning solution that provides due to there is no addition high molecular material, and make final be made
Electrospun fibers there is the performance of micro-nano multistage fiber, distribution of fiber diameters is wider, and due to the spinning that uses of the present invention
Silk liquid natural degree higher so be more advantageous to cell attachment and differentiation effect.
Electrostatic spinning process is carried out using electrostatic spinning reception device, it is preferable that make the fibre object that last spinning obtains
Rationality can be more preferable, and the operating condition of electrostatic spinning is：Syringe injection speed be 1mL/h~4mL/h, syringe needle with receive tablet it
Between distance be 6~10cm, syringe needle adds 6~12kV of positive voltage, and receiver board applies 0.5~1.5kV of negative voltage.This process is using common
Planar metal plate, use international 6~No. 9 flat mouth syringe needles.
Further include that electrospun fibers obtained are used into 2~12h of glutaraldehyde vapor crosslinking after electrostatic spinning.Using
Glutaraldehyde vapor crosslinking can improve the tensile strength of fiber obtained, and it is physical to improve dissolving out capability and swelling character of fiber etc.
Energy.In the present invention, EDC-NHS or genipin cross-linked can also be used other than glutaraldehyde cross-linking can be used.
The electrospinning process of extracellular matrix provided by the invention, due in the process first with fluorine-containing polar solvent with
Appropriate proportioning mixes, then carries out electrostatic spinning as spinning solution in the case where not adding high molecular material, and makes final system
The electrospun fibers obtained have the performance of micro-nano multistage fiber, and distribution of fiber diameters is wider, and used due to the present invention
The natural degree higher of spinning solution is so be more advantageous to the effect of cell attachment and differentiation.And since spinning process is not added with other
Any synthesis macromolecule or natural macromolecular material, high molecular material can be added by overcoming cellular matrix progress electrostatic spinning
Technology prejudice, expanded the processed and applied mode for removing cell matrix materials, and expanded the material selection of electrostatic spinning process
Range.
The present invention also provides a kind of electrospun fibers, use the electrostatic spinning of extracellular matrix provided by the invention
Method is made.Therefore electrospun fibers provided by the invention have micro-nano multistage fiber performance, distribution of fiber diameters compared with
Width, and it is more advantageous to the characteristics of cell adheres to and breaks up.
It is specific to a kind of electrospinning process progress of extracellular matrix provided by the invention below in conjunction with specific embodiment
Explanation.
Embodiment 1
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 30 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property small intestinal mucosa
Lower layer's extracellular matrix.
3g extracellular matrix powder is weighed after sieving, and measures 100mL trifluoroethanols, and trifluoro is added in extracellular matrix powder
In ethyl alcohol, the two is made to carry out being mixed to get mixed solution, it is 2 days that mixed solution, which is stirred mixing time, makes extracellular base
Soluble powder in matter is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, steel ball is added, centrifuge tube is then put into ball milling
In machine -8 degrees Celsius, frequency be 50HZ under conditions of, ball milling 10min.In highly polar solvent in thorough break up cell epimatrix
In insoluble part, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 9 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 1mL/h,
Distance is 10cm between syringe needle and reception tablet, and syringe needle adds positive voltage 6kV, receiver board to apply negative voltage 1.5kV.Static Spinning is made
Silk fiber.
Glutaraldehyde vapor crosslinking 2h is finally used, final products electrospun fibers are obtained.It arrives
Embodiment 2
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 50 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property small intestinal mucosa
Lower layer's extracellular matrix.
4g extracellular matrix powder is weighed after sieving, and measures 100mL hexafluoroisopropanols, and extracellular matrix powder is added six
In fluorine isopropanol, the two is made to carry out being mixed to get mixed solution, it is 7 days that mixed solution, which is stirred mixing time, makes cell
Soluble powder in epimatrix is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, zirconia ball is added, then puts centrifuge tube
Enter in ball mill -12 degrees Celsius, frequency be 80HZ under conditions of, ball milling 60min.In high pole in thorough break up cell epimatrix
Insoluble part in property solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 9 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 4mL/h,
Distance is 6cm between syringe needle and reception tablet, and syringe needle adds positive voltage 12kV, receiver board to apply negative voltage 0.5kV.Static Spinning is made
Silk fiber.
Glutaraldehyde vapor crosslinking 12h is finally used, final products electrospun fibers are obtained.
Embodiment 3
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 40 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property peripheral nerve
Extracellular matrix.
3.2g extracellular matrix powder is weighed after sieving, and measures 100mL hexafluoroisopropanols, and extracellular matrix powder is added
In hexafluoroisopropanol, the two is made to carry out being mixed to get mixed solution, it is 4 days that mixed solution, which is stirred mixing time, is made thin
Soluble powder in extracellular matrix is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, zirconia ball is added, then puts centrifuge tube
Enter in ball mill -11 degrees Celsius, frequency be 60HZ under conditions of, ball milling 20min.In high pole in thorough break up cell epimatrix
Insoluble part in property solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 6 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 2mL/h,
Distance is 7cm between syringe needle and reception tablet, and syringe needle adds positive voltage 8kV, receiver board to apply negative voltage 0.8kV.Static Spinning is made
Silk fiber.
Glutaraldehyde vapor crosslinking 4h is finally used, final products electrospun fibers are obtained.
Embodiment 4
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 50 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property peripheral nerve
Extracellular matrix.
3.4g extracellular matrix powder is weighed after sieving, and measures 100mL hexafluoroisopropanols, and extracellular matrix powder is added
In hexafluoroisopropanol, the two is made to carry out being mixed to get mixed solution, it is 6 days that mixed solution, which is stirred mixing time, is made thin
Soluble powder in extracellular matrix is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, zirconia ball is added, then puts centrifuge tube
Enter in ball mill -10 degrees Celsius, frequency be 75HZ under conditions of, ball milling 30min.In high pole in thorough break up cell epimatrix
Insoluble part in property solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 7 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 2.5mL/
H, distance is 9cm between syringe needle and reception tablet, and syringe needle adds positive voltage 10kV, receiver board to apply negative voltage 1kV.Static Spinning is made
Silk fiber.
Glutaraldehyde vapor crosslinking 10h is finally used, final products electrospun fibers are obtained.
Embodiment 5
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 40 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property peripheral nerve
Extracellular matrix.
3.8g extracellular matrix powder is weighed after sieving, and measures 100mL hexafluoroisopropanols, and extracellular matrix powder is added
In hexafluoroisopropanol, the two is made to carry out being mixed to get mixed solution, it is 5 days that mixed solution, which is stirred mixing time, is made thin
Soluble powder in extracellular matrix is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, steel ball bead is added, is then put into centrifuge tube
In ball mill -9 degrees Celsius, frequency be 70HZ under conditions of, ball milling 40min.Highly polar in thorough break up cell epimatrix
Insoluble part in solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 8 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 3mL/h,
Distance is 8cm between syringe needle and reception tablet, and syringe needle adds positive voltage 11kV, receiver board to apply negative voltage 1.3kV.Static Spinning is made
Silk fiber.
Glutaraldehyde vapor crosslinking 6h is finally used, final products electrospun fibers are obtained.
Embodiment 6
The extracellular matrix for having already passed through Cell extraction is subjected to dozen powder processing using pulverizer, powder is beaten and crosses 40 after the completion
Mesh sieves, and obtains the more uniform extracellular matrix powder of grain size, and the extracellular matrix that the present embodiment is selected is pig source property small intestinal mucosa
Lower layer's extracellular matrix.
3.8g extracellular matrix powder is weighed after sieving, and measures 100mL trifluoroethanols, and extracellular matrix powder is added three
In fluoroethanol, the two is made to carry out being mixed to get mixed solution, it is 3 days that mixed solution, which is stirred mixing time, is made extracellular
Soluble powder in matrix is substantially soluble in solution, insoluble to be divided into even suspension in the solution.
After stirring, mixed solution is placed in the centrifuge tube of 2mL, steel ball bead is added, is then put into centrifuge tube
In ball mill -11 degrees Celsius, frequency be 55HZ under conditions of, ball milling 50min.Highly polar in thorough break up cell epimatrix
Insoluble part in solvent, and suspension is made to homogenize, to obtain to make the better mixed solution of spinning effect.
Mixed solution is not added after the completion of ball milling high molecular material as spinning solution use electrostatic spinning reception device into
Row electrostatic spinning, and using international No. 9 flat mouth syringe needles.The operating condition of electrostatic spinning is：Syringe injection speed is 3.5mL/
H, distance is 8.5cm between syringe needle and reception tablet, and syringe needle adds positive voltage 7kV, receiver board to apply negative voltage 1.4kV.It is made quiet
Electricity spinning fibre.
Glutaraldehyde vapor crosslinking 8h is finally used, final products electrospun fibers are obtained.
Experimental example
The electrospun fibers that embodiment 1 and embodiment 2 obtain are observed into its structure under scanning electron microscope.
The scanning electron microscope (SEM) photograph for the electrospun fibers that embodiment 1 obtains is as shown in Figure 1, the electrostatic spinning that embodiment 2 obtains
The scanning electron microscope (SEM) photograph of fiber is as shown in Figure 2.
By Fig. 1 and Fig. 2, it can be seen that the distribution of fiber diameters of electrospun fibers made from embodiment 1 and embodiment 2
Wider, the characteristic with micro-nano multistage fiber is conducive to cell attachment and differentiation.
In conclusion the electrospinning process of extracellular matrix provided by the invention, due in the process first with it is fluorine-containing
Polar solvent is mixed with appropriate proportioning, then in the case where not adding high molecular material using extracellular matrix as main material as
Spinning solution carries out electrostatic spinning, and final electrospun fibers obtained is made to have the performance of micro-nano multistage fiber, fiber
Diameter wider distribution, and since the natural degree of the spinning solution of the invention used is high, the bioactive ingredients of reservation are more, so more
Be conducive to the effect of cell attachment, differentiation and functionalization.And since spinning process is not added with other any synthesis macromolecules or day
Right high molecular material, the technology prejudice of high molecular material can be added by overcoming cellular matrix progress electrostatic spinning, be expanded
The processed and applied mode of cell matrix materials is gone, and has expanded the material selection range of electrostatic spinning process.And combination will mix
Solution makes suspension by making the insoluble part in extracellular matrix in highly polar solvent thoroughly smash after low temperature ball milling
Homogenization, the physical property for the electrospun fibers that the obtained uniform mixed solution of solute Distribution enables to spinning to obtain is more
It is good.And combine the design parameter of electrostatic spinning process that the physical property that can further increase electrospun fibers is set.Finally exist
The tensile strength that fiber obtained can then be improved after spinning to electrospun fibers glutaraldehyde vapor crosslinking, improves fiber
The physical properties such as dissolving out capability and swelling character.
Electrospun fibers provided by the invention, the performance with micro-nano multistage fiber, distribution of fiber diameters is wider,
Natural degree is high, the characteristics of being more advantageous to cell attachment, differentiation and functionalization.
The foregoing is merely the preferred embodiment of the present invention, are not intended to restrict the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of electrospinning process of extracellular matrix, which is characterized in that operating procedure includes：
The extracellular matrix powder of cell will be gone by 0.03~0.04g:1mL is mixed with fluorine-containing highly polar solvent, obtains mixed solution；
The mixed solution is not added into high molecular material and carries out the obtained electrospun fibers of electrostatic spinning as spinning solution.
2. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that set the mixed solution
In ball mill, high molecular material work is not added after 10~60min of ball milling under conditions of -12~-8 degrees Celsius, 50~80HZ
Electrostatic spinning is carried out for spinning solution.
3. the electrospinning process of extracellular matrix according to claim 2, which is characterized in that by the mixed solution into
After row stirring 2~7 days, it is placed in the ball mill and carries out ball-milling treatment.
4. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that the highly polar solvent packet
Include trifluoroethanol or hexafluoroisopropanol.
5. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that carry out the behaviour of electrostatic spinning
It is as condition：Syringe injection speed is 1mL/h~4mL/h, and distance is 6~10cm between syringe needle and reception tablet, and syringe needle adds
6~12kV of positive voltage, receiver board apply 0.5~1.5kV of negative voltage.
6. the electrospinning process of extracellular matrix according to claim 5, which is characterized in that electrostatic spinning operating process
Using 6~No. 9 flat mouth syringe needles of national standard.
7. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that after electrostatic spinning also
Including electrospun fibers obtained are used 2~12h of glutaraldehyde vapor crosslinking.
8. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that in the cell that will remove cell
Epimatrix powder is mixed with fluorine-containing highly polar solvent further includes before：The extracellular matrix of cell will be gone to carry out beating powder, cross 30~50
Mesh sieves to obtain the extracellular matrix powder for removing cell.
9. the electrospinning process of extracellular matrix according to claim 1, which is characterized in that the extracellular matrix is
Pig source property small intestinal submucosa extracellular matrix or pig source property peripheral nerve extracellular matrix.
10. a kind of electrospun fibers, which is characterized in that using the Static Spinning of the extracellular matrix as described in claim 1-9
Silk method is made.
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Application publication date: 20181012 |