CN108904884A - A kind of preparation method of porcine aorta acellular matrix - Google Patents
A kind of preparation method of porcine aorta acellular matrix Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3641—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 site of application in the body
- A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The present invention provides a kind of preparation methods of porcine aorta acellular matrix, include the following steps：Porcine aorta is placed in 2~6h of freezing in low concentration of detergent buffer under vacuum conditions by step 1；After freezing, the porcine aorta water-bath after freezing is dissolved for step 2；Step 3 repeats step 1 and step 2 1~10 time, porcine aorta is carried out thawing；Porcine aorta after thawing is placed in low concentration of detergent buffer by step 4, and 28~96h of processing concussion in 30~50 DEG C of shaking tables；Step 5, with aseptic deionized water to 12~48h of porcine aorta concussion cleaning after concussion；And step 6, after 48~96h of porcine aorta concussion cleaning after being cleaned with phosphate solution to aseptic deionized water, obtain the acellular matrix of porcine aorta cell, wherein low concentration of detergent buffer includes Tris buffer, lauryl sodium sulfate and NaTDC.
Description
Technical field
The present invention relates to field of tissue engineering technology, and in particular to a kind of preparation method of porcine aorta acellular matrix.
Background technique
It is not only developed country with the vascular conditions that aortic aneurysm, vascular dissection, Atherosclerosis turn to representative, still
One of primary lethal lesion of developing country.Surgical replacement lesion vessels are the final hands for the treatment of vascular diseases
Section, thus need various vascular replacement materials.In addition, increasing year by year with Incidence of esophageal cancer, artificial esophagus displacement material
Demand it is also more and more.
Although homologue displacement is ideal displacement material, source is few, there are ethics limitations.And currently with life
The development of object science and technology has developed a variety of high-molecular biologic synthetic materials as artificial organ displacement material.For example, PET
(Polyethylene terephthalate, Dacron) and ePTFE (Expanded polytetra-
Fluoroethylene), polyurethane (Polyurethane) is because it is with preferable biomechanical property and due to internal compatibility
It is widely used in cardiovascular surgical procedure.But synthetic material also has limitation, such as synthesising biological material in clinical application
It is to increase mortality an important factor for leading to graft failure with natural tissues mismatch.In addition, lasting chronic inflammation is anti-
The risks such as caused transplanting tissue endometrial hyperplasia, chronic infection are answered to increase the incidence that decays of transplanting later period.
Therefore, a kind of similar with Normal aorta, oesophagus substitute in institutional framework and function is researched and developed to closing weight
It wants.Based on this reason, cell porcine aorta is taken off due to it substantially, under microscope and in physiological function is being similar to human aorta
It receives more and more attention.Also, some researches show that porcine aorta functionally can replace oesophagus.
De- cell is to reduce transplanting tissue immunogenicity in Tissue Engineering Study, retain graft engineering three-dimensional tissue structures, life
Object mechanical property, the important method of bioactivity, also, method for removing cells has been successfully applied to heart valve, prostate, angle
In the Tissue Engineering Studies such as film, heel string.It is cell free to realize that the prior art generally handles porcine aorta using detergent
Purpose, although this method can obtain good de- cell effect, the de- cell porcine aorta that obtains by this method
Matrix often remaining toxicity, and its biomechanical property is also destroyed.In addition, a large number of studies show that cell free pig master
The extra-cellular matrix structure of artery is fine and close, and therefore, cell is difficult endogenous growth when applied in host, the weight in host
It moulds ineffective.
Summary of the invention
The present invention is to carry out to solve the above-mentioned problems, and it is an object of the present invention to provide a kind of porcine aorta acellular matrix
Preparation method.
The present invention provides a kind of preparation methods of porcine aorta acellular matrix, have the feature that, including following
Step：Porcine aorta is placed in 2~6h of freezing in low concentration of detergent buffer under vacuum conditions by step 1；Step 2,
After freezing, the porcine aorta water-bath after freezing is dissolved；Step 3 repeats step 1 and step 2 1~10 time, by pig master
Artery carries out thawing；Porcine aorta after thawing is placed in low concentration of detergent buffer by step 4, and
28~96h of processing concussion in 30~50 DEG C of shaking tables；Step 5 shakes cleaning to the porcine aorta after concussion with aseptic deionized water
12~48h；And step 6,48~96h of porcine aorta concussion cleaning after being cleaned with phosphate solution to aseptic deionized water
Afterwards, the acellular matrix of porcine aorta cell is obtained, wherein low concentration of detergent buffer includes Tris buffer, dodecane
Base sodium sulphate and NaTDC, the concentration range of Tris buffer are 8~12mmol/L, and pH value is 7.4~7.8,12
Sodium alkyl sulfate is 0.1%~0.5%W/V in the volume fraction range of Tris buffer, and NaTDC is in Tris buffer
Volume fraction range be 0~0.8%W/V.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, in step 2, the temperature of water-bath is 30~50 DEG C.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, the concentration of Tris buffer is 10mmol/L, PH 7.6.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, in step 1, the cooling time of porcine aorta is 4~5h.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, in step 5, the concussion scavenging period of deionized water is 24~36h.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, the concentration of lauryl sodium sulfate is 0.1~0.5mmol/L.
In the preparation method of porcine aorta acellular matrix provided by the invention, it can also have the feature that：Its
In, in step 6, the concussion scavenging period of phosphate solution is 72h.
The action and effect of invention
The preparation method of related porcine aorta acellular matrix according to the present invention, because with pig master in the preparation method
Artery is research object, is freezed using low concentration of detergent as buffer, and then water-bath is dissolved, and porcine aorta is passed through
Be placed in detergent buffer after thawing and shake, finally respectively with deionized water and phosphate solution to concussion after
Porcine aorta carry out concussion cleaning, the acellular matrix of porcine aorta can be obtained, so, preparation method of the invention obtains
Acellular matrix be in micropore spline structure, good mechanical performance, and non-toxic, immunogenicity is low, and calcification rate is low, can be used for blood
The transplanting of the clinical tissues engineering such as pipe, oesophagus, and remoldability is good in vivo.
In addition, due to this preparation method be using porcine aorta as research object, this preparation method it is low in cost,
The valuable medical resource for saving several hundred million members is engineering blood vessel, the clinical application of oesophagus and the optimization of acellular matrix
Research and development provide experimental basis and pointer.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Embodiment is closed to be specifically addressed the preparation method of porcine aorta acellular matrix of the present invention.
The preparation method of porcine aorta acellular matrix mainly includes the following steps that：
Porcine aorta is placed in 2~6h of freezing in low concentration of detergent buffer under vacuum conditions by step 1, low dense
Spending detergent buffer includes：Tris buffer, lauryl sodium sulfate and NaTDC.The concentration model of Tris buffer
It encloses for 8~12mmol/L, pH value is 7.4~7.8, and lauryl sodium sulfate is in the volume fraction range of Tris buffer
0.1%~0.5%W/V, NaTDC are 0~0.8%W/V in the volume fraction range of Tris buffer.In the present embodiment
In, the proportion of buffer, lauryl sodium sulfate and NaTDC is：The concentration of Tris buffer is 10mmol/L, PH
It is 7.6, the deoxycholic acid of 0.1~0.4g lauryl sodium sulfate and 0.1~0.5g is added in the Tris buffer of every 100mL
Sodium.Cooling time of the porcine aorta in low concentration of detergent buffer is 4~5h.
Step 2 after freezing, the porcine aorta after freezing is placed in the water-bath that temperature is 30~50 DEG C and is dissolved.
Step 3 repeats step 1 and step 2 1~10 time, porcine aorta is carried out thawing.
Porcine aorta after thawing is taken out from low concentration of detergent buffer, is then placed by step 4
In dirty agent buffer, and 28~96h of processing concussion in 30~50 DEG C of shaking tables.
Step 5, with aseptic deionized water to 12~48h of porcine aorta concussion cleaning after concussion.In the present embodiment,
Aseptic deionized water is 24~36h to the concussion scavenging period of porcine aorta, and the preferably concussion time is for 24 hours.
Step 6 obtains after 48~96h is cleaned in the porcine aorta concussion after being cleaned with phosphate solution to aseptic deionized water
To the acellular matrix of porcine aorta.In the present embodiment, phosphate solution is 72h to the concussion scavenging period of porcine aorta.
With the vacuum freeze thawing in the present embodiment-low concentration lauryl sodium sulfate (Vacuum-freeze-thawing-low
Concentration SDS, VLS) acellular matrix of obtained porcine aorta is handled for experimental group, with high concentration dodecyl
The acellular matrix for the porcine aorta that sodium sulphate (High concentration SDS, HCS) processing obtains is control group, to upper
It states two groups of acellular matrixes progress structures and performance measurement, obtained result is as follows：
(1) vacuum freeze thawing-low concentration lauryl sodium sulfate method can effectively remove porcine aorta cell DNA, the company of being formed
Continuous micropore spline structure
Pathology H&E dyeing shows that compared with normal porcine aorta, VLS method and HCS can effectively remove cell and cell
Fragment.
DNA quantitative detection result is：The DNA content of HCS group is 818.4 ± 226.2ng/mg wet tissue weight；VLS group
DNA content is 497.5 ± 323.6ng/mg wet tissue weight.DNA quantitative detection then shows that pig master can be effectively reduced in VLS and HCS
DNA content in arterial tissue, and the DNA content in VLS processing tissue handles tissue (P significantly lower than HCS<0.05).
Scanning electron microscope show VLS processing organize the formation of continuous micropore sample as a result, and HCS processing tissue can also be formed it is micro-
Hole spline structure, but hole wall is broken.
(2) vacuum freeze thawing-low concentration lauryl sodium sulfate method is able to maintain porcine aorta biomechanical property and three-dimensional
Structure
Biomechanical property testing result is as shown in table 1：
Table 1
As shown in table 1, long axis biomechanical analysis shows compared with normal porcine aorta, and VLS handles porcine aorta most
Big load, maximum stress and elasticity modulus are without obvious statistical significance (P>0.05).But HCS processing but significantly reduces pig
Maximum load, maximum stress and the elasticity modulus (P of aorta<0.05).
After horizontal axis biomechanical analysis shows VLS processing, maximum load, maximum stress and the springform of porcine aorta
Amount rises.HCS processing but significantly reduces the maximum load (P of porcine aorta<0.05).
Elastic force collagen staining shows that the porcine aorta extra-cellular matrix structure of VLS processing is complete, and HCS destroys pig actively
Arteries and veins extracellular matrix, the fracture of partial region collagen elastic force.
(3) porcine aorta no cytotoxicity handled by vacuum freeze thawing-low concentration lauryl sodium sulfate method
Extracting cytotoxicity detection shows that compared with normal incubation medium handles cell, VLS processing extracting matrix can significantly promote
Into the growth of huve cell and mescenchymal stem cell, but HCS processing extracting matrix leads to a large amount of Apoptosis, bad
Extremely, growth inhibition.The growth ability of VLS group huve cell is the 1.5 ± 0.1 of normal incubation medium Cell growth ability
Times, and the growth ability of HCS group cell is only 0.3 ± 0.1 times of normal incubation medium Cell growth ability.VLS group mesenchyma is dry
The growth ability of cell is 1.1 ± 0.1 times of normal incubation medium Cell growth ability, and the growth ability of HCS group cell is only
0.6 ± 0.3 times of normal incubation medium Cell growth ability.VLS group is compared with HCS group has apparent statistical significance (P<
0.05)。
Exposing cell toxicity detection shows that the huve cell that VLS processing porcine aorta co-cultures and mesenchyma are dry
The upgrowth situation of cell is good, but a large amount of downright bad, apoptosis of cell co-cultured with HCS processing porcine aorta, floating spherical in shape.
(4) in vivo, the calcification and inflammation of vacuum freeze thawing-low concentration lauryl sodium sulfate method processing porcine aorta
Response light
The porcine aorta subcutaneous rat of processing is embedded, takes out detection calcification and inflammatory reaction in particular point in time.As a result
Show at the 28th day, the calcification degree of VLS processing (19.2 ± 5.4/ unit) porcine aorta be substantially less than HCS group (31.8 ±
12.5) it, compares with statistical significance (P for two groups<0.05).
Inflammatory reaction detection then shows early stage, and HCS processing porcine aorta leads to lasting acute inflammatory reaction, peak period
(231.2 ± 139.3/high power field of view) on day 3, and VLS processing group acute inflammatory reaction is light, is just within the 3rd day.
Middle and later periods, the visible T cell chronic lymphocytic infiltration based on CD4 and CD8 in two groups of grafts.At the 28th day, in HCS group
The quantity of CD4 and cd8 cell is respectively 90.0 ± 23.3/high power field of view, 165.8 ± 65.3/high power field of view；And
The quantity of CD4 and cd8 cell in VLS group are then respectively 61.2 ± 13.4/high power field of view, 57.8 ± 30.8/high power
The mirror visual field.VLS group is compared with HCS group has apparent statistical significance (P<0.05).
(5) in vivo, the blood vessel and tissue of vacuum freeze thawing-low concentration lauryl sodium sulfate method processing porcine aorta
Remodeling is significant
Immunohistochemical staining the result shows that, at 28 days, myofibroblast is uniformly distributed in VLS processing porcine aorta
In each layer of porcine aorta；Although HCS processing porcine aorta center also have myofibroblast infiltration (28 days, 37.7 ± 19.3/
High power field of view), but quantity is substantially less than the quantity (P of VLS processing group (28 days, 127.5 ± 20.3/high power field of view)<
0.05).Myofibroblast is the important cells for secreting collagen, and consistent with myofibroblast distribution, collagen staining shows
Around in VLS processing porcine aorta and middle section has newborn collagen to be formed.
When blood vessel immunohistochemistry results show 28 days, new vessels are uniformly distributed in pig in VLS processing porcine aorta
Each layer of aorta；HCS processing porcine aorta (28 days, 15.8 ± 12.4/high power field of view) is although also there are new vessels in center
It is formed, but quantity is substantially less than the quantity (P of VLS processing group (28 days, 39.3 ± 11.9/high power field of view)<0.05).
The action and effect of embodiment
According to the preparation method of the porcine aorta acellular matrix in above-described embodiment, because with pig master in the preparation method
Artery is research object, is freezed using low concentration of detergent as buffer, and then water-bath is dissolved, and porcine aorta is passed through
Be placed in detergent buffer after thawing and shake, finally respectively with deionized water and phosphate solution to concussion after
Porcine aorta carry out concussion cleaning, the acellular matrix of porcine aorta can be obtained, so, preparation method of the invention obtains
Acellular matrix be in micropore spline structure, good mechanical performance, and non-toxic, immunogenicity is low, and calcification rate is low, can be used for blood
The transplanting of the clinical tissues engineering such as pipe, oesophagus, and remoldability is good in vivo.
In addition, due to this preparation method be using porcine aorta as research object, this preparation method it is low in cost,
The valuable medical resource for saving several hundred million members is engineering blood vessel, the clinical application of oesophagus and the optimization of acellular matrix
Research and development provide experimental basis and pointer.
In addition, in the above-described embodiments, because the low concentration of detergent buffer used is low concentration dodecyl sulphate
Sodium buffer, includes Tris buffer, lauryl sodium sulfate and NaTDC in the buffer, so, it is of the invention
The cell and nuclear material of porcine aorta tissue can be effectively removed in preparation method, reduces internal inflammatory reaction and calcium deposition.
In addition, the preparation method in the present embodiment can promote porcine aorta to organize the formation of continuous micropore spline structure, it should
Micropore spline structure can promote detergent intrusion organization internal removal cell, and then can reduce the use concentration of detergent, have
Effect retains the three-dimensional structure and biomechanical property of porcine aorta tissue, and significantly reduces the residual toxicity of detergent.
Moreover, the micropore spline structure of the acellular matrix of the porcine aorta obtained through the foregoing embodiment can promote
Fibroblast and vascular endothelial cell infiltrative growth, to promote pipe newborn with collagen.
Above embodiment is preferred case of the invention, the protection scope being not intended to limit the invention.
Claims (6)
1. a kind of preparation method of porcine aorta acellular matrix, which is characterized in that include the following steps：
Porcine aorta is placed in 2~6h of freezing in low concentration of detergent buffer under vacuum conditions by step 1；
After freezing, the porcine aorta water-bath after freezing is dissolved for step 2；
Step 3 repeats step 1 and step 2 1~10 time, and the porcine aorta is carried out thawing；
The porcine aorta after thawing is placed in low concentration of detergent buffer by step 4, and at 30~50 DEG C
28~96h of processing concussion in shaking table；
Step 5, with aseptic deionized water to 12~48h of porcine aorta concussion cleaning after concussion；And
Step 6 obtains pig after 48~96h is cleaned in the porcine aorta concussion after being cleaned with phosphate solution to aseptic deionized water
The acellular matrix of aorta cells,
Wherein, the low concentration of detergent buffer includes Tris buffer, lauryl sodium sulfate and NaTDC,
The concentration range of the Tris buffer is 8~12mmol/L, and pH value is 7.4~7.8,
The lauryl sodium sulfate is 0.1%~0.5%W/V in the volume fraction range of the Tris buffer,
The NaTDC is 0~0.8%W/V in the volume fraction range of the Tris buffer.
2. the preparation method of porcine aorta acellular matrix according to claim 1, it is characterised in that：
Wherein, in step 2, the temperature of the water-bath is 30~50 DEG C.
3. the preparation method of porcine aorta acellular matrix according to claim 1, it is characterised in that：
Wherein, the concentration of the Tris buffer is 10mmol/L, PH 7.6.
4. the preparation method of porcine aorta acellular matrix according to claim 1, it is characterised in that：
Wherein, in step 1, the cooling time of the porcine aorta is 4~5h.
5. the preparation method of porcine aorta acellular matrix according to claim 1, it is characterised in that：
Wherein, in step 5, the concussion scavenging period of the deionized water is 24~36h.
6. the preparation method of porcine aorta acellular matrix according to claim 1, it is characterised in that：
Wherein, in step 6, the concussion scavenging period of the phosphate solution is 72h.
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| CN112190758A (en) * | 2020-09-17 | 2021-01-08 | 扬州大学 | Method for rapidly preparing acellular tracheal stent |
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