CN108261564A - De- extracellular matrix and its preparation method and application - Google Patents
De- extracellular matrix and its preparation method and application Download PDFInfo
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- CN108261564A CN108261564A CN201611262064.4A CN201611262064A CN108261564A CN 108261564 A CN108261564 A CN 108261564A CN 201611262064 A CN201611262064 A CN 201611262064A CN 108261564 A CN108261564 A CN 108261564A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention discloses a kind of preparation method of de- extracellular matrix, extracellular matrix and the de- extracellular matrix are taken off made from the preparation method of the de- extracellular matrix in the application for preparing medical instruments field.A kind of preparation method of de- extracellular matrix, includes the following steps：Tissue precursor is obtained after being pre-processed to the histoorgan of mammal；It is virus inactivated processing, DNA removings processing, de- cell processing and ungrease treatment respectively to tissue precursor, obtains de- extracellular matrix precursor；Serial dehydration processing and crosslinking Treatment are carried out successively to de- extracellular matrix precursor, finally sizing is dried to obtain de- extracellular matrix, and serial dehydration processing is：The de- extracellular matrix precursor is immersed in successively in the organic solution that concentration successively increases.The preparation method of this de- extracellular matrix handles tissue precursor by serial dehydration, and the ordered structure of the collagenous fibres of final de- extracellular matrix obtained can be caused not to be destroyed.
Description
Technical field
The present invention relates to medical material field, more particularly to a kind of de- extracellular matrix and its preparation method and application.
Background technology
The cell epimatrix material using animal tissue as raw material based on organizational engineering principle is the main direction of development.Carefully
Extracellular matrix is organic by the complexity of a variety of macromolecular substances such as ingredients such as collagen, non-collagen glycoprotein, elastin laminin structure
Three-dimensional overall structure, existence and activity for various cells provide suitable place and microenvironment, organization of regulation control organ dysfunction.Institute
Using extracellular matrix as ideal tissue renovation material, clinic is widely used for, including pericardium, pleura, diaphragm, peritonaeum
With the cell epimatrix materials such as submucous layer of small intestine.
Self and allosome tissue source is extremely limited, and xenogenesis is (such as：Pig, ox, horse) de- extracellular matrix just become
One big theme of current soft tissue repair technical research.There are many kinds of the preparation methods of de- extracellular matrix, it is therefore an objective to can go
Except ingredients such as cell all in tissue, fat, and retain the integrality of collagen component and structure to the maximum extent.
De- cell technique and virus inactivation technology are the main technique and technological difficulties of de- cell epimatrix material, it is desirable that complete
Virus, cell and the animal sources DNA ingredients of full removal animal tissue, at the same the complete ingredient for retaining natural de- extracellular matrix and
Three-dimensional structure.The method type for preparing de- extracellular matrix is various, but cannot remove completely mostly all animal derived DNA into
Point, and time-consuming, needs, using a variety of organic and surfactant equal solvent, on the one hand to result in acellular matrix material
The destruction of active constituent, on the other hand, hazardous solvent residual can lead to cytotoxicity, stimulation and immune response, so as to influence group
Knit repairing effect.And the hydrophily of de- extracellular matrix and applicating property be not strong mostly, rate of water absorption is slow, and film is not thin enough and soft,
So as to not adapt to the reparation of various surface profile tissues.
The ordered structure that the collagenous fibres of extracellular matrix are taken off made from the preparation method of existing de- extracellular matrix meets with
To destruction, so as to affect subsequent applications.
Invention content
Based on this, it is necessary to provide it is a kind of can be to avoid the de- extracellular matrix that the ordered structure of collagenous fibres is destroyed
Extracellular matrix and application are taken off made from preparation method and the preparation method of the de- extracellular matrix.
A kind of preparation method of de- extracellular matrix, includes the following steps：
Tissue precursor is obtained after being pre-processed to the histoorgan of mammal；
It is virus inactivated processing, DNA removings processing, de- cell processing and ungrease treatment respectively to the tissue precursor,
De- extracellular matrix precursor is obtained, wherein, the viral inactivation treatment, DNA removings processing, the de- cell processing and institute
Stating the operation order of ungrease treatment can mutually replace；
Serial dehydration processing and crosslinking Treatment are carried out successively to the de- extracellular matrix precursor, finally sizing is dried to obtain
De- extracellular matrix, wherein, the serial dehydration processing is：Concentration is immersed in successively successively to the de- extracellular matrix precursor
In increased organic solution, the time impregnated every time is 10min~60min.
In one embodiment, the serial dehydration, which is handled, is：The de- extracellular matrix precursor is immersed in matter successively
Measure in the organic solution that percentage concentration is 25%, 50%, 75%, 90% and 100%, time for impregnating every time for 10min~
60min, wherein, the solute of the organic solution is ethyl alcohol or acetone.
In one embodiment, the operation that the histoorgan to the mammal is pre-processed is：With machinery
It is clear with pure water after method carries out the histoorgan of the mammal in preliminary removal fat, muscle and other attached organs
Wash clean.
In one embodiment, the histoorgan of the mammal is the histoorgan of pig, ox or horse.
In one embodiment, the pericardium of the histoorgan of the mammal including mammal, pleura, diaphragm,
Peritonaeum and submucous layer of small intestine.
In one embodiment, the histoorgan of the mammal is the peritonaeum of pig.
In one embodiment, the viral inactivation treatment is：The tissue precursor is immersed in inactivation of virus solution
1h~10h is virus inactivated processing, and the inactivation of virus solution is the peroxide that mass percentage concentration is 0.1%~5%
Solution or the inactivation of virus solution are the ethyl alcohol of the peroxide containing 0.1wt%~5wt% and 20wt%~70wt%
Mixed solution；
The DNA removings are handled：The tissue precursor is immersed in 1h in aqueous slkali~for 24 hours, then be immersed in acid solution
Middle 1h~12h；
The de- cell, which is handled, is：The tissue precursor is immersed in the egg that mass percentage concentration is 0.05%~0.5%
6h~48h in white enzyme solutions, then it is immersed in 6h~48h in the NaCl solution of a concentration of 0.5M~3M；
The ungrease treatment is：The tissue precursor is immersed in degreasing organic matter, it is 1 time~5 times to impregnate number, often
The time of secondary immersion is 1h~for 24 hours；
The crosslinking Treatment is：Under conditions of temperature is 90 DEG C~150 DEG C, to by the described de- of serial dehydration processing
Extracellular matrix precursor progress high-temperature vacuum crosslinking 1h~for 24 hours；
Alternatively, the crosslinking Treatment is：Matter will be immersed in by the de- extracellular matrix precursor of serial dehydration processing
Measure 1h~for 24 hours in the cross-linking agent solution that percentage concentration is 0.25%~3%.
In one embodiment, the peroxide solutions in hydrogenperoxide steam generator and peracetic acid soln at least
It is a kind of；
The peroxide is selected from least one of hydrogen peroxide and Peracetic acid；
The aqueous slkali is selected from sodium carbonate liquor, sodium bicarbonate solution, sodium hydroxide solution or potassium hydroxide solution, described
The mass percentage concentration of aqueous slkali is 0.5%~4%；
The acid solution is selected from least one of hydrochloric acid and acetum, and the mass percentage concentration of the acid solution is
0.05%~2%；
The protease is selected from least one of trypsase, pepsin and neutral proteinase；
The degreasing organic matter is selected from least one of n-hexane, isopropanol, ethylene glycol and ether；
It is molten that the cross-linking agent solution is selected from glutaraldehyde solution, carbodiimides solution, genipin solution and epoxide
At least one of liquid.
A kind of de- extracellular matrix is prepared using the preparation method of above-mentioned de- extracellular matrix.
Above-mentioned de- extracellular matrix, in the application for preparing medical instruments field.
The preparation method of this de- extracellular matrix, handled by viral inactivation treatment, DNA removings, the processing of de- cell and
Ungrease treatment eliminates the immunogenicity of tissue precursor, then has by what tissue precursor to be immersed in concentration successively increases successively
Serial dehydration in machine object solution, then crosslinking Treatment and qualitative drying obtain de- extracellular matrix.Group is handled by serial dehydration
Precursor is knitted, the ordered structure of the collagenous fibres of final de- extracellular matrix obtained can be caused not to be destroyed.In addition, simultaneously by
In with orderly collagen fiber structure, this de- extracellular matrix have excellent flexibility, good biocompatibility and
Self-bone grafting ability, while rate of water absorption is fast and water suction does not expand, and has a good application prospect.
Description of the drawings
Fig. 1 is the flow chart of the preparation method of the de- extracellular matrix of an embodiment；
Fig. 2 is the electron scanning micrograph of the shiny surface obtained for preparing de- extracellular matrix of embodiment 1；
Fig. 3 is the electron scanning micrograph of the rough surface obtained for preparing de- extracellular matrix of embodiment 1；
Fig. 4 is that the inverted microscope obtained for preparing de- extracellular matrix after hematoxylin eosin staining of embodiment 1 shines
Piece；
Fig. 5 be embodiment 1 it is obtained prepare in de- extracellular matrix implantation in rabbit daughter after a week slice by hematoxylin-she
Inverted microscope photo after red colouring.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment pair
The specific embodiment of the present invention is described in detail.Many details are elaborated in the following description in order to fully manage
The solution present invention.But the invention can be embodied in many other ways as described herein, those skilled in the art can
To do similar improvement without violating the connotation of the present invention, therefore the present invention is not limited by following public specific implementation
System.
As shown in Figure 1, the preparation method of the de- extracellular matrix the invention discloses an embodiment, including walking as follows
Suddenly：
S10, tissue precursor is obtained after being pre-processed to the histoorgan of mammal.
The operation pre-processed to the histoorgan of mammal is：With Mechanical Method to the histoorgan of mammal into
After the preliminary removal fat of row, muscle and other attached organs, cleaned up and (preferably cleaned 3~5 times) with pure water.Its
In, other attached organs indicate the organ except pericardium, pleura, diaphragm, peritonaeum and submucous layer of small intestine.
Preferably, the histoorgan of mammal is the histoorgan of pig, ox or horse.
Preferably, pericardium, pleura, diaphragm, peritonaeum and small intestine of the histoorgan of mammal including mammal glue
Film lower floor.
Particularly preferred, the histoorgan of mammal is the peritonaeum of pig.
The tissue precursor that S10 is obtained can be stored in -20 DEG C it is spare.
S20, the tissue precursor that S10 is obtained is virus inactivated respectively processing, DNA removings processing, the processing of de- cell and
Ungrease treatment obtains de- extracellular matrix precursor.
In S20, viral inactivation treatment, DNA removings processing, de- cell processing and the operation order of ungrease treatment can be mutual
It replaces.
In S20, viral inactivation treatment is：Tissue precursor is immersed in 1h~10h in inactivation of virus solution (preferably 3h)
It is virus inactivated processing.
Inactivation of virus solution is the peroxide solutions that mass percentage concentration is 0.1%~5% (preferably 2%).Peroxide
Compound solution is selected from least one of hydrogenperoxide steam generator and peracetic acid soln.
Alternatively, inactivation of virus solution is peroxide and 20wt% containing 0.1wt%~5wt% (preferably 2wt%)
The mixed solution of the ethyl alcohol of~70wt% (preferably 50wt%).Peroxide in hydrogen peroxide and Peracetic acid at least
It is a kind of.
In S20, DNA removings processing is：Tissue precursor is immersed in 1h in aqueous slkali~(preferably 12h), then impregnate for 24 hours
1h~12h (the preferably 3h) in acid solution.
Aqueous slkali is selected from sodium carbonate liquor, sodium bicarbonate solution, sodium hydroxide solution or potassium hydroxide solution, aqueous slkali
Mass percentage concentration is 0.5%~4% (preferably 2%).
Acid solution is selected from least one of hydrochloric acid and acetum, the mass percentage concentration of acid solution for 0.05%~
2% (preferably 0.5%).
In S20, de- cell processing is：Tissue precursor is immersed in mass percentage concentration (is preferably for 0.05%~0.5%
0.25%) 6h~48h (preferably for 24 hours) in protein enzyme solution, then it is immersed in a concentration of 0.5M~3M's (preferably 1M)
6h~48h (preferably 12h) in NaCl solution.
Protease is selected from least one of trypsase, pepsin and neutral proteinase.
In S20, ungrease treatment is：Tissue precursor is immersed in degreasing organic matter, it is 1 time~5 times to impregnate number, every time
The time of immersion is 1h~for 24 hours.
Preferably, ungrease treatment is：Tissue precursor is immersed in n-hexane, impregnate number be 2 times, impregnate every time when
Between be 6h；Then it is immersed in isopropanol, it is 2 times to impregnate number, and the time impregnated every time is 12h.
Degreasing organic matter is selected from least one of n-hexane, isopropanol, ethylene glycol and ether.
S30, serial dehydration processing and crosslinking Treatment are carried out successively to the de- extracellular matrix precursor that S20 is obtained, it is finally fixed
Type is dried to obtain de- extracellular matrix.
In S30, serial dehydration processing is：The organic matter for being immersed in concentration successively to taking off extracellular matrix precursor and successively increasing
In solution, the time impregnated every time is 10min~60min.
Preferably, serial dehydration, which is handled, is：By de- extracellular matrix precursor be immersed in successively mass percentage concentration for 25%,
50%th, in 75%, 90% and 100% organic solution, the time impregnated every time is (preferably each for 10min~60min
20min), wherein, the solute of organic solution is ethyl alcohol or acetone.
Crosslinking Treatment is：Under conditions of temperature is 90 DEG C~150 DEG C, to the de- extracellular base by serial dehydration processing
Matter precursor progress high-temperature vacuum crosslinking 1h~for 24 hours.
Alternatively, crosslinking Treatment is：It is dense by quality percentage is immersed in by the de- extracellular matrix precursor of serial dehydration processing
Spend for 0.25%~3% 1h in the cross-linking agent solution of (preferably 2.5%)~for 24 hours.
Cross-linking agent solution is selected from glutaraldehyde solution, carbodiimides solution, genipin solution and epoxide (preferably
At least one of butanediol diglycidyl ether) solution.
Preferably, cross-linking agent solution is that the butanediol diglycidyl ether that pH is 5.5, mass percentage concentration is 2.5% is molten
Liquid.
The dry operation of sizing is：Corresponding size and the mold of shape are designed according to product requirement, serial dehydration will be passed through
The de- extracellular matrix precursor of processing is fixed on mold, and the material being fixed on mold is dried or dried, dry
De- extracellular matrix is obtained after the completion.
Drying can be freeze-drying (18h~for 24 hours) or vacuum drying (1h~for 24 hours, preferably 3h).
Obtained de- extracellular matrix is packed in ten thousand grades of dust proof workshops, is packed using inside and outside vacuum formed box, plastic uptake
Box is defended strong paper using spy and is sealed, and is sterilized after the completion of packaging using 60Coradiation or electron beam irradiation.
The preparation method of this de- extracellular matrix, handled by viral inactivation treatment, DNA removings, the processing of de- cell and
Ungrease treatment eliminates the immunogenicity of tissue precursor, then has by what tissue precursor to be immersed in concentration successively increases successively
Serial dehydration in machine object solution, then crosslinking Treatment and qualitative drying obtain de- extracellular matrix.Group is handled by serial dehydration
Precursor is knitted, the ordered structure of the collagenous fibres of final de- extracellular matrix obtained can be caused not to be destroyed.In addition, simultaneously by
In with orderly collagen fiber structure, this de- extracellular matrix have excellent flexibility, good biocompatibility and
Self-bone grafting ability, while rate of water absorption is fast and water suction does not expand, and has a good application prospect.
The invention also discloses a kind of de- extracellular matrixs, are prepared into using the preparation method of above-mentioned de- extracellular matrix
It arrives.
Using the animal sources DNA less residues of de- extracellular matrix prepared by the above method, and completely remain natural fine
Three-dimensional structure, the immunogenicity of extracellular matrix are low, flexibility and applicating property are good, rate of water absorption is fast and water suction does not expand, it is good to have
Good biological safety and biocompatibility, can adjust growth, shape, metabolism, migration, increment and the differentiation of cell, and then
Organization of regulation control and organ dysfunction are ideal tissue renovation materials.
This de- extracellular matrix has excellent flexibility, good biocompatibility and self-bone grafting ability, inhales simultaneously
Water speed rate is fast and water suction does not expand, and has a good application prospect, can be applied to prepare medical instruments field.
It is specific embodiment below.
Embodiment 1
The peritoneal tissues of fresh pig are taken, pig peritonaeum is carried out with Mechanical Method tentatively to remove fat, muscle and attached other
Organ after removing subsidiary other compositions, is made pig peritonaeum thickness as 0.3mm~1.0mm, is cleaned 4 times with pure water, be stored in-
20 DEG C spare.
Pretreated pig peritonaeum is immersed in successively in the NaOH solution of 2wt% and handles 12h, it is clear with pure water after processing
It washes 3~5 times, then handles 2h with the HCl solution of a concentration of 0.5wt%, cleaned 4 times with pure water after processing, to remove animal
Organize most DNA.
DNA is removed that treated pig peritonaeum is immersed in 3h in the hydrogenperoxide steam generator of 2wt% and is virus inactivated processing.
Pig peritonaeum after inactivation treatment is immersed in the neutral protein enzyme solutions of 0.25wt% for 24 hours, pure water rinsing is simultaneously soaked
Bubble carries out de- cell processing for 24 hours in the hypertonic saline of 1M.
Pig peritonaeum is sequentially placed into n-hexane and aqueous isopropanol and carries out degreasing, n-hexane processing time is for 24 hours, repeats
Processing 1 time；Isopropanol processing time is 12h, is reprocessed 2 times.
Pig peritonaeum is sequentially placed into ethanol solution and carries out serial dehydration, the mass percentage concentration of Gradient elution using ethanol is
25%th, 50%, 75%, 90%, 100%, the time being dehydrated every time is 20min.
Pig peritonaeum acellular matrix is immersed in glutaraldehyde and is crosslinked, a concentration of 0.25wt% of glutaraldehyde, crosslinking
Time is 1h.
Design corresponding size and the mold of shape are required according to different product, material is fixed on mold, and will consolidate
It is freeze-dried due to the material on mold, drying time 20h.
It is packed in ten thousand grades of dust proof workshops, is packed using inside and outside vacuum formed box, vacuum formed box is defended strong paper using spy and carried out
Sealing, packaging use 60Coradiation after the completion.
It is observed extracellular matrix is taken off made from embodiment 1 under scanning electron microscope (SEM), obtains Fig. 2~Fig. 3.
Extracellular matrix is taken off made from embodiment it can be seen from Fig. 2~Fig. 31 has orderly collagen fiber structure, light
Sliding surface is fine and close, and rough surface is loose fibre structure, is conducive to adherency and growth of the later stage osteoblast in rough surface.
Extracellular matrix de- made from embodiment 1 is carried out to see under inverted microscope after hematoxylin-eosin (HE) dyes
It examines, obtains Fig. 4.
As seen from Figure 4, being taken off in extracellular matrix made from embodiment 1 does not have cell fragment residual, and can see
Observe fibre structure.
It is sliced being taken off in extracellular matrix implantation in rabbit daughter made from embodiment 1 after a week, by hematoxylin-eosin (HE)
It is observed under inverted microscope after dyeing, obtains Fig. 5.
As seen from Figure 5, it will take off in extracellular matrix (collagem membrane) implantation in rabbit daughter made from embodiment 1 and do not have after a week
There is inflammatory reaction, and sticking performance is good, de- extracellular matrix (collagem membrane) does not expand.
Embodiment 2
The peritoneal tissues of fresh ox are taken, ox peritonaeum is carried out with Mechanical Method tentatively to remove fat, muscle and attached other
Organ after removing subsidiary other compositions, is made ox peritonaeum thickness as 0.3mm~1.0mm, is cleaned 5 times with pure water, be stored in-
20 DEG C spare.
Pretreated ox peritonaeum is immersed in successively in the NaOH solution of 2wt% and handles 10h, it is clear with pure water after processing
It washes 5 times, then handles 4h with the HCl solution of a concentration of 0.25wt%, cleaned 5 times with pure water after processing, to remove animal groups
Knit most DNA.
DNA is removed that treated ox peritonaeum is immersed in the mixed solution of the Peracetic acid of 1wt% and the ethyl alcohol of 50wt%
Middle 2h is virus inactivated processing.
12h in the trypsin solution of 0.25wt% is immersed in the ox peritonaeum after inactivation treatment, pure water rinsing is simultaneously impregnated
12h carries out de- cell processing in the hypertonic saline of 2M.
Ox peritonaeum is sequentially placed into n-hexane and aqueous isopropanol and carries out degreasing, n-hexane processing time is 6h, is repeated
Processing 2 times；Isopropanol processing time is for 24 hours, reprocesses 1 time.
Ox peritonaeum is sequentially placed into acetone soln and carries out serial dehydration, the mass percentage concentration of Gradient elution using ethanol is
25%th, 50%, 75%, 90%, 100%, the time being dehydrated every time is 20min.
Ox peritonaeum acellular matrix is immersed in carbodiimides and is crosslinked, a concentration of 0.5wt% of glutaraldehyde is handed over
Join the time for for 24 hours.
Design corresponding size and the mold of shape are required according to different product, material is fixed on mold, and will consolidate
It is dried in vacuo due to the material on mold, drying time 12h.
It is packed in ten thousand grades of dust proof workshops, is packed using inside and outside vacuum formed box, vacuum formed box is defended strong paper using spy and carried out
Sealing, packaging use 60Coradiation after the completion.
Embodiment 3
The pericardial tissue of fresh ox is taken, bovine pericardium is carried out with Mechanical Method tentatively to remove fat, muscle and attached
Other organs after removing subsidiary other compositions, are made bovine pericardium thickness as 0.3mm~1.0mm, are cleaned 3 times with pure water, stored up
It is spare in the presence of -20 DEG C.
Pretreated bovine pericardium is immersed in successively in the NaOH solution of 4wt% and handles 6h, pure water is used after processing
Then cleaning 3 times handles 6h with the HCl solution of a concentration of 1wt%, is cleaned 3 times with pure water after processing, to remove animal tissue
Most DNA.
DNA removes to treated that bovine pericardium is immersed in the Peracetic acid of 2wt% and the alcohol mixed solution of 70wt%
Middle 1h is virus inactivated processing.
12h in 0.5% pepsin solution is immersed in the bovine pericardium after inactivation treatment, pure water rinsing is simultaneously immersed in
12h carries out de- cell processing in the hypertonic saline of 0.5M.
Bovine pericardium is sequentially placed into n-hexane, ethylene glycol ethyl ethers ethereal solution and carries out degreasing, n-hexane processing time is
12h is reprocessed 2 times；Ethylene glycol ethyl ether processing time is 6h, is reprocessed 1 time.
Bovine pericardium is sequentially placed into acetone soln and carries out serial dehydration, the mass percentage concentration of Gradient elution using ethanol is
25%th, 50%, 75%, 90%, 100%, the time being dehydrated every time is 10min.
Design corresponding size and the mold of shape are required according to different product, material is fixed on mold, and will consolidate
Vacuum high-temperature crosslinking and drying are carried out due to the material on mold, drying time is for 24 hours, crosslinking temperature is set as 100 DEG C.
It is packed in ten thousand grades of dust proof workshops, is packed using inside and outside vacuum formed box, vacuum formed box is defended strong paper using spy and carried out
Sealing, packaging use 60Coradiation after the completion.
Embodiment 4
The pleura of fresh pig is taken, the pleura of pig is carried out with Mechanical Method tentatively to remove fat, muscle and attached its
His organ, after removing subsidiary other compositions, is cleaned 4 times with pure water, be stored in -20 DEG C it is spare.
Pretreated pig pleura is immersed in successively in the NaOH solution of 3wt% and handles 5h, it is clear with pure water after processing
It washes 4 times, then handles 5h with the HCl solution of a concentration of 0.1wt%, cleaned 4 times with pure water after processing, to remove animal tissue
Most DNA.
DNA removes to treated that pig pleura is immersed in 2h in the Peracetic acid of 1wt% and 20% alcohol mixed solution
It is virus inactivated processing.
12h in the pepsin solution of 0.5wt% is immersed in the pig pleura after inactivation treatment, pure water rinsing is simultaneously immersed in
12h carries out de- cell processing in the hypertonic saline of 0.5M.
Pig pleura is put into aqueous isopropanol and carries out degreasing, isopropanol processing time is 12h, is reprocessed 4 times.
Pig pleura is sequentially placed into ethanol solution and carries out serial dehydration, the mass percentage concentration of Gradient elution using ethanol is
25%th, 50%, 75%, 90%, 100%, the time being dehydrated every time is 30min.
Pig pleura acellular matrix is immersed in epoxide and is crosslinked, epoxide shrinks for butanediol two
Glycerin ether, a concentration of 2.5wt% of crosslinker solution, pH value 5.5.
Design corresponding size and the mold of shape are required according to different product, material is fixed on mold, and will consolidate
It is freeze-dried due to the material on mold, drying time 18h.
It is packed in ten thousand grades of dust proof workshops, is packed using inside and outside vacuum formed box, vacuum formed box is defended strong paper using spy and carried out
Sealing, packaging use 60Coradiation after the completion.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that those of ordinary skill in the art are come
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of preparation method of de- extracellular matrix, which is characterized in that include the following steps：
Tissue precursor is obtained after being pre-processed to the histoorgan of mammal；
It is virus inactivated processing, DNA removings processing, de- cell processing and ungrease treatment respectively to the tissue precursor, obtains
De- extracellular matrix precursor, wherein, the viral inactivation treatment, the DNA removings are handled, the de- cell is handled and described de-
The operation order of fat processing can be replaced mutually；
Serial dehydration processing and crosslinking Treatment are carried out successively to the de- extracellular matrix precursor, finally sizing is dried to obtain de- thin
Extracellular matrix, wherein, the serial dehydration processing is：Concentration is immersed in successively to the de- extracellular matrix precursor to successively increase
Organic solution in, time for impregnating every time is 10min~60min.
2. the preparation method of de- extracellular matrix according to claim 1, which is characterized in that the serial dehydration processing
For：It is 25%, 50%, 75%, 90% and 100% that the de- extracellular matrix precursor is immersed in mass percentage concentration successively
In organic solution, the time impregnated every time is 10min~60min, wherein, the solute of the organic solution is ethyl alcohol or third
Ketone.
3. the preparation method of de- extracellular matrix according to claim 1, which is characterized in that described to the mammal
The operation that is pre-processed of histoorgan be：The histoorgan of the mammal is carried out with Mechanical Method tentatively to go grease removal
After fat, muscle and other attached organs, cleaned up with pure water.
4. the preparation method of the de- extracellular matrix according to claim 1 or 3, which is characterized in that the mammal
Histoorgan is the histoorgan of pig, ox or horse.
5. the preparation method of the de- extracellular matrix according to claim 1 or 3, which is characterized in that the mammal
Histoorgan includes pericardium, pleura, diaphragm, peritonaeum and the submucous layer of small intestine of mammal.
6. the preparation method of the de- extracellular matrix according to claim 1 or 3, which is characterized in that the mammal
Histoorgan is the peritonaeum of pig.
7. the preparation method of de- extracellular matrix according to claim 1, which is characterized in that the viral inactivation treatment
For：The tissue precursor is immersed in 1h~10h in inactivation of virus solution and is virus inactivated processing, the inactivation of virus solution
For mass percentage concentration be 0.1%~5% peroxide solutions or the inactivation of virus solution be containing 0.1wt%~
The mixed solution of the peroxide of 5wt% and the ethyl alcohol of 20wt%~70wt%；
The DNA removings are handled：The tissue precursor is immersed in 1h in aqueous slkali~for 24 hours, then be immersed in 1h in acid solution
~12h；
The de- cell, which is handled, is：The tissue precursor is immersed in the protease that mass percentage concentration is 0.05%~0.5%
6h~48h in solution, then it is immersed in 6h~48h in the NaCl solution of a concentration of 0.5M~3M；
The ungrease treatment is：The tissue precursor is immersed in degreasing organic matter, it is 1 time~5 times to impregnate number, is soaked every time
The time of bubble is 1h~for 24 hours；
The crosslinking Treatment is：Under conditions of temperature is 90 DEG C~150 DEG C, to the de- cell by serial dehydration processing
Epimatrix precursor progress high-temperature vacuum crosslinking 1h~for 24 hours；
Alternatively, the crosslinking Treatment is：Quality hundred will be immersed in by the de- extracellular matrix precursor of serial dehydration processing
1h in point a concentration of 0.25%~3% cross-linking agent solution~for 24 hours.
8. the preparation method of de- extracellular matrix according to claim 7, which is characterized in that the peroxide solutions choosing
From at least one of hydrogenperoxide steam generator and peracetic acid soln；
The peroxide is selected from least one of hydrogen peroxide and Peracetic acid；
The aqueous slkali is selected from sodium carbonate liquor, sodium bicarbonate solution, sodium hydroxide solution or potassium hydroxide solution, the alkali soluble
The mass percentage concentration of liquid is 0.5%~4%；
The acid solution is selected from least one of hydrochloric acid and acetum, and the mass percentage concentration of the acid solution is 0.05%
~2%；
The protease is selected from least one of trypsase, pepsin and neutral proteinase；
The degreasing organic matter is selected from least one of n-hexane, isopropanol, ethylene glycol and ether；
The cross-linking agent solution is in glutaraldehyde solution, carbodiimides solution, genipin solution and epoxide solution
At least one.
9. a kind of de- extracellular matrix, which is characterized in that the de- extracellular matrix is used such as any one of claim 1~8
The preparation method of the de- extracellular matrix is prepared.
10. de- extracellular matrix according to claim 9, in the application for preparing medical instruments field.
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