CN109621010A - A kind of Acellular cartilaginous matrix and preparation method thereof - Google Patents
A kind of Acellular cartilaginous matrix and preparation method thereof Download PDFInfo
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- CN109621010A CN109621010A CN201811544160.7A CN201811544160A CN109621010A CN 109621010 A CN109621010 A CN 109621010A CN 201811544160 A CN201811544160 A CN 201811544160A CN 109621010 A CN109621010 A CN 109621010A
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Classifications
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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
- A61L27/3612—Cartilage, synovial fluid
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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
-
- 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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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- Biomedical Technology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
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- Oral & Maxillofacial Surgery (AREA)
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Abstract
The present invention relates to articular cartilage technical fields, more particularly to a kind of Acellular cartilaginous matrix and preparation method thereof, cartilaginous tissue is passed sequentially through into Hypotonic treatment, trypsin treatment, detergent-treatment and nucleic acid enzymatic treatment, cartilage matrix is made, Acellular cartilaginous matrix prepared by the present invention is capable of the three-dimensional structure of cartilage-preserving cellular matrix to the maximum extent itself, with good mechanical performance, and it does not need additional crosslink agent and carries out physics or chemical crosslinking, immunogenicity is low, toxicity is low, and preparation method is simple, cost is relatively low, suitable for industrial production.
Description
Technical field
The present invention relates to articular cartilage technical fields, and in particular to a kind of Acellular cartilaginous matrix and preparation method thereof.
Background technique
Articular cartilage is the important feature of human synovial, and the major function of articular cartilage is to protect skeletal joint face from mill
Damage.Because being distributed in articular cartilage without blood vessel, lymph and cartilage, and cartilage cell's height is broken up, so that articular cartilage itself is repaired
Ability is extremely limited, and articular cartilage is once destroyed, and the pathological change of generation is often irreversible, and regeneration is extremely difficult, and
Cartilage damage seriously affects the normal walking and daily routines of people, and long-term cartilage damage is also the main reason for leading to deformity of limbs
One of.
De- cell technology is the method for removing the histocyte reservation extracellular matrix of immunogenicity, the extracellular base of cartilage
Not only contain the several functions compositions such as bioactive molecule, collagen, non-collagen glycoprotein, elastin laminin, glycosaminoglycan in matter,
It can promote cartilaginous tissue reparation, control migration, proliferation, differentiation and the metabolism of cell, and due to the extracellular matrix of cartilage
For tridimensional network, be conducive to sticking, being proliferated for cell, provide good growing environment for cell growth, take off cell technology
Have become very important research direction and research contents in cartilaginous tissue reparation.
Commonly used method for removing cells mainly has Mechanical Method, enzyme digestion, chemical detergent method etc. both at home and abroad at present, however
Be used alone or be used in combination these methods it is cell free during be easy to appear cell antigenicity removal be not thorough, cell
The problem of three-dimensional structure of epimatrix is by destroying influences cartilage so that cytotrophy supply and metabolic waste discharge are difficult
Repairing effect even results in repair of cartilage failure.In the prior art, in order to thoroughly remove cartilage cell, main use first will
The method that cartilage smashes or is ground into particle, this results in needing repeatedly to be centrifuged and be cleaned multiple times in subsequent processing, no
The destruction for only having aggravated the three-dimensional structure of extracellular matrix causes Acellular cartilaginous matrix material mechanical performance to be deteriorated, is mechanical
Strength reduction.
For example, a kind of Chinese patent literature CN108066823A discloses cell Acellular cartilaginous matrix and its preparation side
Method is specifically comprised the following steps: (1) to be ground into after cartilage particles in hypotonic solution and is shaken with the speed of 50-300rpm/min
24-72h.(2) and ionic surface active agent vibrates 3-6h under conditions of temperature is 37-40 DEG C, revolving speed is 50-300rpm/min
Obtain the second cartilage.(3) it shakes under conditions of temperature is 37-40 DEG C, revolving speed is 50-300rpm/min with pancreatin and nuclease
Swing 0.5-2h.(4) it after first irradiating 1-3h under ultraviolet light, then immerses and reacts 2-6h in crosslinking agent.Above-mentioned Acellular cartilaginous matrix
Preparation method in, although by physics and chemical crosslinking can largely improve Acellular cartilaginous matrix mechanics it is strong
Degree, keeps it more stable, however, implanting a period of time the problem of having toxicity and its residual because of additional crosslink agent itself
It is easily immunoreacted afterwards.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that overcoming Acellular cartilaginous matrix mechanical performance in the prior art
Difference, immunogenicity and the high defect of toxicity, to provide a kind of Acellular cartilaginous matrix and preparation method thereof.
The present invention provides a kind of preparation method of Acellular cartilaginous matrix, by cartilaginous tissue pass sequentially through Hypotonic treatment,
Cartilage matrix is made in trypsin treatment, detergent-treatment and nucleic acid enzymatic treatment.
In Hypotonic treatment, cartilaginous tissue is added in hypotonic solution and is incubated for, is incubated for 15-30h at room temperature；
Preferably, the Tris-Hcl solution that the hypotonic solution is 10mM and pH is 7-8.
In trypsin treatment, cartilaginous tissue is added in trypsin solution, the heating water bath 6-24h at 37 DEG C；
Preferably, the trypsin solution is the PBS solution containing 0.10-0.50% (m/v) trypsase.
In detergent-treatment, cartilaginous tissue is added in detergent solution, oscillation treatment at being 15-25 DEG C in temperature
15-30h；
Preferably, the detergent solution is the Tris-Hcl solution of the 8-10mM containing 0.05-0.3%SDS.
In nucleic acid enzymatic treatment, by cartilaginous tissue be added buffered cell liquid in, then be added 30-60U/ml DNA enzymatic and
The RNA enzyme of 0.5-4U/ml, the heating water bath 6-20h at 35-40 DEG C；
Preferably, the buffered cell liquid is the Tris-Hcl solution of the magnesium chloride containing 5-30mM, the Tris-Hcl solution
Concentration be 30-60mM, pH 7-8.
It further include that multigelation processing is carried out to cartilaginous tissue before Hypotonic treatment；
Preferably, in multigelation processing, cartilaginous tissue is placed at -70~-80 DEG C and freezes 2-4h, is subsequently placed in
Thaw 1-6h at 15-25 DEG C, and the number of multigelation is 2-6 times.
It further include neutral protein enzymatic treatment before nucleic acid enzymatic treatment；
Preferably, in neutral protein enzymatic treatment, cartilaginous tissue is added to the life of the neutral proteinase containing 300-700U/L
It manages in salt water, the heating water bath 4-12h at 30-40 DEG C；
It preferably, further include α -1,3- galactoside enzyme solutions processing, in α -1,3- half after neutral protein enzymatic treatment
In lactoside enzymatic treatment, cartilaginous tissue is added to α -1 of the g/mL of μ containing 10-40, in the PBS solution of 3- galactosidase, in 25-
At 37 DEG C, impregnate 1-6 hours.
It further include sterilization treatment after nucleic acid enzymatic treatment；
Preferably, in sterilization treatment, cartilaginous tissue is added in thimerosal and impregnates 1-6h, the thimerosal be containing
The PBS solution of 0.05-0.4vt% acetic acid and 0.05-0.4wt% sodium hypochlorite, or
PBS solution containing 0.05-0.4vt% acetic acid and 0.05-0.4vt%84 thimerosal.
Cartilaginous tissue further include before Hypotonic treatment before cleaning treatment；In Hypotonic treatment, trypsin treatment, detergent
It further include intermediate cleaning treatment after processing and nucleic acid enzymatic treatment；Cleaning treatment after further including after nucleic acid enzymatic treatment；
Preferably, in preceding cleaning treatment, cartilaginous tissue is added in physiological saline and is impregnated, be then added in ultrapure water and surpass
Sound cleaning, then with ultrapure water continual rinsing；
Preferably, in intermediate cleaning treatment, cartilaginous tissue is added in ultrapure water and is cleaned by ultrasonic, then continued with ultrapure water
It rinses；
Preferably, in rear cleaning treatment, cartilaginous tissue is added in sterile PBS and is impregnated.
The present invention provides Acellular cartilaginous matrixes made from the preparation method described in one kind.
Technical solution of the present invention has the advantages that
1. the preparation method of Acellular cartilaginous matrix provided by the invention, the cartilaginous tissue after cleaning successively passes through hypotonic place
Reason, trypsin treatment and detergent-treatment finally pass through nucleic acid enzymatic treatment, and Acellular cartilaginous matrix is made.Wherein, by low
The osmosis of osmometer solution makes moisture constantly penetrate cell membrane, makes cell membrane swelling fracture, promotes trypsin treatment and decontamination
De- cell solution in agent processing enters cell through cell membrane, so as to improve trypsin treatment and detergent-treatment
De- cell effect, specifically, cartilaginous tissue pass through trypsin digestion and cell film surface and intracellular immune protein, effectively drop
Low immunogenicity retains collagen and glycosaminoglycan, then by the cell component in detergent removal cartilaginous tissue, destroys cell
Membrane phospholipid and adipose membrane, thoroughly remove antigen and immune complex, and the DNA in nucleus can be removed finally by nucleic acid enzymatic treatment
And RNA；The present invention does not enhance the de- cell of trypsin treatment and detergent-treatment merely with first hypotonic solution processing
Effect, and de- cell effect also can be improved by the Combined Treatment of trypsin treatment and detergent, reduce detergent
Dosage, be effectively improved because detergent residual or detergent-treatment it is excessive caused by Acellular cartilaginous matrix cytotoxicity or
The enhancing of person's immunogenicity, while can also improve trypsin treatment and excessively cause to the excessive of collagen and elastin laminin
The case where digestion causes the three-dimensional structure disorder of Acellular cartilaginous matrix matrix and mechanical strength to reduce.Thus, the present invention
The Acellular cartilaginous matrix of preparation is capable of the three-dimensional structure of cartilage-preserving cellular matrix to the maximum extent itself, has good machine
Tool performance, and do not need additional crosslink agent and carry out physics or chemical crosslinking, immunogenicity is low, toxicity is low, and preparation side
Method is simple, and cost is relatively low, suitable for industrial production.
2. the preparation method of Acellular cartilaginous matrix provided by the invention, cartilaginous tissue is placed in hypotonic solution and is incubated for,
It is incubated for 15-30h at room temperature, so that the moisture in hypotonic solution is slowly entered cartilage cell in a mild condition, avoids hypotonic solution
The integrality for destroying the structure of cartilage cell epimatrix, improves the stability of mechanical performance, further, using 8-10mM and pH
It is hypotonic solution for the Tris-Hcl solution of 7-8, can further improves hypotonic solution treatment process to Acellular cartilaginous matrix
The influence of the integrality of structure improves Acellular cartilaginous matrix mechanical performance, reduces toxicity.
3. cartilaginous tissue is added in trypsin solution the preparation method of Acellular cartilaginous matrix provided by the invention,
The heating water bath 6-24h at 37 DEG C can further increase the integrality of the structure of chondrocyte matrix, improve de- cellular cartilage
Matrix mechanical performance and stability.And the present invention uses and cartilaginous tissue is added in detergent solution, is 15-25 DEG C in temperature
Lower oscillation treatment 15-30h has that reinforces to go using the Tris-Hcl solution of the 8-10mM containing 0.05-0.3%SDS as detergent
Dirty effect guarantees that de- cell is complete.
4. the preparation method of Acellular cartilaginous matrix provided by the invention, by the way that buffered cell liquid first is added in cartilaginous tissue
In, then be added 30-60U/ml DNA enzymatic and 0.5-4U/ml RNA enzyme, the nuclease of high concentration in buffer solution gradually
Dispersion, thoroughly digests the nucleic acid in cartilaginous tissue, and the immunogenicity of Acellular cartilaginous matrix is effectively reduced, and reduces between batch
The difference of toxicity improves the stability of product quality, and by the way of heating water bath, enters nuclease in a mild condition
Cartilaginous tissue digests DNA and RNA, reduces destruction to Acellular cartilaginous matrix, improve Acellular cartilaginous matrix mechanical performance and
Stability.
5. the preparation method of Acellular cartilaginous matrix provided by the invention further includes to cartilaginous tissue before Hypotonic treatment
Multigelation processing is carried out, multigelation processing can make extracellular matrix more loose, be conducive to subsequent moisture and de- cell
The entrance of solution improves de- cell effect, immunogenicity and toxicity is further decreased, by the way that cartilaginous tissue is placed in -70~-80
2-4h is freezed at DEG C, is subsequently placed at 15-25 DEG C the 1-6h that thaws, multigelation 1-6 times can retain cell to the maximum extent
While epimatrix three-dimensional structure, de- cell effect is improved.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the HE of the Acellular cartilaginous matrix in experimental example 4 under microscope and the cartilaginous tissue without taking off cell processing
Colored graph, wherein A is cartilaginous tissue (500 μm), and B is cartilaginous tissue (100 μm), and C is Acellular cartilaginous matrix (500 μm), and D is
For Acellular cartilaginous matrix (100 μm)；There are a large amount of blue dyes in A and B, is contaminated in C and D without indigo plant；
Fig. 2 is the Acellular cartilaginous matrix in experimental example 5 under microscope and the cartilaginous tissue without taking off cell processing
DAPI colored graph, wherein A is cartilaginous tissue, and B is Acellular cartilaginous matrix；There is stronger blue-fluorescence in A, without blue-fluorescence in B；
Fig. 3 is the ɑ-of the Acellular cartilaginous matrix in experimental example 6 under microscope and the cartilaginous tissue without taking off cell processing
Gal immunohistochemical staining figure, wherein A is cartilaginous tissue, and B is Acellular cartilaginous matrix；There is stronger red fluorescence in A, in B
Redfree fluorescence；
Fig. 4 is changing for Acellular cartilaginous matrix in experimental example 7 under microscope and the cartilaginous tissue without taking off cell processing
Good tri- color of Masson dye figure, wherein A is cartilaginous tissue, and B is Acellular cartilaginous matrix；There are stronger blue and blue brown, blue in A
For collagenous fibres, blue brown is nucleus；Only relatively strong blue in B；
Fig. 5 is the II of the Acellular cartilaginous matrix in experimental example 8 under microscope and the cartilaginous tissue without taking off cell processing
Collagen Type VI immunohistochemistry dye figure, wherein A is cartilaginous tissue, and B is Acellular cartilaginous matrix；There are stronger red fluorescence and blue in A
Fluorescence, only red fluorescence in B.
Specific embodiment
There is provided following embodiments is for a better understanding of the present invention, it is not limited to the preferred forms are not right
The contents of the present invention and protection scope are construed as limiting, anyone under the inspiration of the present invention or by the present invention with other existing skills
The feature of art is combined and any and identical or similar product of the present invention for obtaining, all falls within protection scope of the present invention
Within.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Fresh cartilage tissue below, will be with cartilage after obtaining pig costal cartilage using adult healthy pig costal cartilage as material
The removals such as connected muscle, fascia are clean, the final cartilaginous tissue for obtaining transparence.
Embodiment 1
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in physiological saline, impregnates, then by cartilaginous tissue
It takes out and is added in ultrapure water, be cleaned by ultrasonic through ultrasonic washing instrument, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in sterilized 10L Tris-Hcl solution, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, concentration 10mM.
(4) trypsin treatment: cartilaginous tissue is put into trypsin solution, the heating water bath 8h at 37 DEG C.Wherein,
Trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, and the volume of trypsin solution is 10L.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at 20 DEG C
Oscillation treatment 72h, wherein the Tris-Hcl liquor capacity containing 0.1% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save it is stand-by.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 2
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 1kg fresh is added in physiological saline, impregnates, then takes cartilaginous tissue
It is added in ultrapure water, is cleaned by ultrasonic through ultrasonic washing instrument out, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 2h, then thaw 6h at room temperature, repeatedly
Freeze thawing 6 times.
(3) Hypotonic treatment: cartilaginous tissue is added in sterilized Tris-Hcl solution, 15h is incubated at room temperature.Its
In, the pH of Tris-Hcl solution is 8, volume 10L, concentration 10mM.
(4) trypsin treatment: by cartilaginous tissue after ultrapure water cleans, being put into trypsin solution, at 37 DEG C
Heating water bath 6h.Wherein, trypsin solution is the PBS solution containing 0.20% (m/v) trypsase, the body of trypsin solution
Product is 10L.
(5) detergent-treatment: by cartilaginous tissue after ultrapure water cleans, it is put into the 10mM's containing 0.3% (m/v) SDS
In Tris-Hcl, oscillation treatment 15h at room temperature, wherein the volume of the Tris-Hcl solution containing 0.3% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 700U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 12h.
(7) nucleic acid enzymatic treatment: by cartilaginous tissue after ultrapure water cleans, contain the 5mM magnesium chloride and pH for being put into 10L are 8
30mM Tris-Hcl solution in, the DNA enzymatic of 30U/ml and the RNA enzyme of 4U/ml is added, the heating water bath 6h at 37 DEG C.
(8) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 37 DEG C, impregnate 6 hours.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.4vt% acetic acid and 0.05vt%,
6h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 3
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 500g fresh is added in physiological saline, impregnates, then by cartilaginous tissue
It takes out and is added in ultrapure water, be cleaned by ultrasonic through ultrasonic washing instrument, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -70 DEG C and freezes 4h, then thaw at 30 DEG C 1h, repeatedly
Freeze thawing 2 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 8mM, is incubated at room temperature
30h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) trypsin treatment: by cartilaginous tissue after ultrapure water cleans, being put into trypsin solution, at 37 DEG C
Heating water bath is for 24 hours.Wherein, trypsin solution is the PBS solution containing 0.30% (m/v) trypsase, trypsin solution
Volume is 10L.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.05% (m/v) SDS, at room temperature
Oscillation treatment 30h, wherein the volume of the Tris-Hcl solution containing 0.05% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 300U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 4h.
(8) it nucleic acid enzymatic treatment: by cartilaginous tissue after ultrapure water cleans, is put into containing 30mM magnesium chloride and pH is 7.0
In the Tris-Hcl solution of 60mM, the DNA enzymatic of 60U/ml and the RNA enzyme of 0.5U/ml is added, the heating water bath 20h at 25 DEG C.Its
In, the volume of Tris-Hcl solution is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.05vt% acetic acid and 0.4vt%,
2h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 4
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in physiological saline, impregnates, then by cartilaginous tissue
It takes out and is added in ultrapure water, be cleaned by ultrasonic through ultrasonic washing instrument, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, at 15 DEG C, is turned
20h is vibrated under conditions of fast 200rpm/min.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) trypsin treatment: cartilaginous tissue is put into trypsin solution, at 37 DEG C, revolving speed 200rpm/min
Under conditions of vibrate 8h.Wherein, trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, trypsin solution
Volume be 10L.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the volume of the Tris-Hcl solution containing 0.1% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 5
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in physiological saline, impregnates, then by cartilaginous tissue
It takes out and is added in ultrapure water, be cleaned by ultrasonic through ultrasonic washing instrument, then with ultrapure water continual rinsing.
(2) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(3) trypsin treatment: cartilaginous tissue is put into trypsin solution, the heating water bath 8h at 37 DEG C.Wherein,
Trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, and the volume of trypsin solution is 10L.
(4) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the volume of the Tris-Hcl solution containing 0.1% (m/v) SDS is 10L.
(5) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(6) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(7) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save it is stand-by.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(6), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 6
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) trypsin treatment: cartilaginous tissue is put into 10L trypsin solution, the heating water bath 8h at 37 DEG C.Its
In, trypsin solution is the PBS solution containing 0.25% (m/v) trypsase.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of 10mM of the 10L containing 0.1% (m/v) SDS, room temperature
Lower oscillation treatment 72h.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Embodiment 7
The preparation method of Acellular cartilaginous matrix, includes the following steps: in the present embodiment
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in ultrapure water, impregnates, then takes cartilaginous tissue
It is added in ultrapure water, is cleaned by ultrasonic through ultrasonic washing instrument out, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) trypsin treatment: cartilaginous tissue is put into trypsin solution, the heating water bath 8h at 37 DEG C.Wherein,
Trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, and the volume of trypsin solution is 10L.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the Tris-Hcl liquor capacity containing 0.1% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS containing 0.2vt% acetic acid, impregnates 3h at room temperature, is carried out
Sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Comparative example 1
The preparation method of Acellular cartilaginous matrix, includes the following steps: in this comparative example
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in ultrapure water, impregnates, then takes cartilaginous tissue
It is added in ultrapure water, is cleaned by ultrasonic through ultrasonic washing instrument out, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the Tris-Hcl liquor capacity containing 0.1% (m/v) SDS is 10L.
(5) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(6) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(7) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(8) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save it is stand-by.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(7), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Comparative example 2
The preparation method of Acellular cartilaginous matrix, includes the following steps: in this comparative example
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in ultrapure water, impregnates, then takes cartilaginous tissue
It is added in ultrapure water, is cleaned by ultrasonic through ultrasonic washing instrument out, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(4) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the Tris-Hcl liquor capacity containing 0.1% (m/v) SDS is 10L.
(5) trypsin treatment: cartilaginous tissue is put into trypsin solution, the heating water bath 8h at 37 DEG C.Wherein,
Trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, and the volume of trypsin solution is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Comparative example 3
The preparation method of Acellular cartilaginous matrix, includes the following steps: in this comparative example
(1) cleaning treatment before: the cartilaginous tissue for taking 800g fresh is added in ultrapure water, impregnates, then takes cartilaginous tissue
It is added in ultrapure water, is cleaned by ultrasonic through ultrasonic washing instrument out, then with ultrapure water continual rinsing.
(2) multigelation is handled: cartilaginous tissue being first placed at -80 DEG C and freezes 3h, then thaw 4h at room temperature, repeatedly
Freeze thawing 4 times.
(3) trypsin treatment: the cartilaginous tissue after multigelation is put into trypsin solution, the water-bath at 37 DEG C
Heat 8h.Wherein, trypsin solution is the PBS solution containing 0.25% (m/v) trypsase, and the volume of trypsin solution is
10L。
(4) Hypotonic treatment: cartilaginous tissue is added in the Tris-Hcl solution of sterilized 10mM, is incubated at room temperature
20h.Wherein, the pH of Tris-Hcl solution is 8, volume 10L.
(5) detergent-treatment: cartilaginous tissue is put into the Tris-Hcl of the 10mM containing 0.1% (m/v) SDS, at room temperature
Oscillation treatment 72h, wherein the Tris-Hcl liquor capacity containing 0.1% (m/v) SDS is 10L.
(6) cartilaginous tissue is put into the physiological saline of the neutral proteinase containing 400U/L by neutral protein enzymatic treatment, and 37 DEG C
Lower heating water bath 8h.
(7) cartilaginous tissue, is added the α -1 for containing 20 μ g/mL, 3- galactosidase by the processing of α -1,3- galactoside enzyme solutions
PBS solution in, at 35 DEG C, impregnate 2 hours.
(8) nucleic acid enzymatic treatment: the Tris-Hcl that cartilaginous tissue is put into the 50mM that containing 10mM magnesium chloride and pH is 7.5 is molten
In liquid, the DNA enzymatic of 50U/ml and the RNA enzyme of 1U/ml is added, the heating water bath 12h at 37 DEG C.Wherein, Tris-Hcl solution
Volume is 10L.
(9) sterilization treatment: cartilaginous tissue being added in the PBS of 84 thimerosals containing 0.2vt% acetic acid and 0.2vt%,
3h is impregnated at room temperature, carries out sterilization treatment.
(10) cleaning treatment afterwards: and then being added in sterile PBS and impregnate, and encapsulates, be stored in -20 DEG C of refrigerator save to
With.
It further include intermediate cleaning treatment after every the end of the step wherein in step (2)-(9), intermediate cleaning treatment is using ultrapure
Water cleans cartilaginous tissue, is subsequently placed in ultrasonic washing instrument and cleans.
5 rounds are repeated using the preparation method, the Acellular cartilaginous matrix of 5 different batches is prepared.
Experimental example 1
The DNA residual quantity of Acellular cartilaginous matrix is able to reflect its de- cell effect, also can by reducing DNA residual quantity
Enough reduce the immunogenicity of Acellular cartilaginous matrix.
Acellular cartilaginous matrix obtained and not cell free cartilage group in Example 1-6 and comparative example 1-3 respectively
25mg is knitted, respectively as test group, control group and blank control group, is cut into small pieces respectively, according to " Chinese Pharmacopoeia " 2015 editions
DNA in the Acellular cartilaginous matrix of fluorescence colour measurement different batches in three 3407 Residual exogenous DNA amount measuring methods
As a result residual quantity see the table below shown.
DNA residual quantity result in the Acellular cartilaginous matrix of different batches in table 1 embodiment 1-6 and comparative example 1-3
Experimental example 2
Acellular cartilaginous matrix obtained and not cell free cartilaginous tissue in Example 1-6 and comparative example 1-3, point
Not Zuo Wei test group, control group and blank control group, its cytotoxicity is measured respectively using mtt assay, using serum-free DMEM
Culture medium is prepared into 12 groups of Acellular cartilaginous matrix leaching liquors according to GB/T1688.12-2008 standard, presses as extraction medium
According to GB/T14233.2 standard, 1 week old new zealand white rabbit femur bone marrow is taken, using Density Gradient Centrifugation Isolation and culture BMSCs
Cell culture, then cell of the inoculation in logarithmic growth phase in 96 orifice plates, cell implantation concentrations are 7 × 104A/ml.If
Blank group is set, 100 μ l cell suspensions are added in remaining every hole.At 37 DEG C, contain 5%CO2Incubator in after overnight incubation, test group
It is separately added into the leaching liquor of 100 μ l, 100 μ l culture solutions are added in control group, and every group sets 8 parallel holes.After cultivating 48h, 20 μ are added
LMTT (5mg/mlPBS) solution is put into incubator after continuing to cultivate 4h, supernatant is carefully sucked out, and it is sub- that 150 μ l diformazans are added
Sulfone, concussion 10min to bottom hole purple crystal are completely dissolved.Each hole absorbance OD value is measured with enzyme linked immunological microplate reader, is calculated
The opposite proliferation rate of each group Acellular cartilaginous matrix treated BMSCs cell.
The Acellular cartilaginous matrix cytotoxicity result of different batches in table 2 embodiment 1-6 and comparative example 1-3
Experimental example 3
By Acellular cartilaginous matrix made from embodiment 1-6 and comparative example 1-3 and not cell free cartilaginous tissue, respectively
As test group, control group and blank control group, plate is respectively prepared, it is soft to take off cell using stretching strength determination instrument measurement each group
The tensile strength of bone matrix, records maximum pull when breaking, and maximum pull is in the sectional area of cartilage matrix, as stretches
Intensity is shown in Table 3.
The tensile strength result of the Acellular cartilaginous matrix of different batches in table 3 embodiment 1-6 and comparative example 1-3
From table 1-3 it is found that compared with comparative example 1-3, the tensile strength of the Acellular cartilaginous matrix in embodiment 1-6,
Cell opposite proliferation rate obviously increases, DNA residual quantity, the coefficient of variation of DNA residual quantity, the coefficient of variation of cell opposite proliferation rate
There is significant decrease with the coefficient of variation of tensile strength, wherein the DNA residual quantity of Acellular cartilaginous matrix is 24.86-
100.45ng/mg, cell opposite proliferation rate are 59.3-99.5%, and cytotoxicity grade is not more than 2 grades, tensile strength 2.31-
3.84MPa illustrates that Acellular cartilaginous matrix cell removal effect of the invention is reliable, and immunogenicity is low, toxicity is low, cartilage cell
It is high that the three-dimensional structure of matrix itself retains integrality；And the DNA residual quantity of Acellular cartilaginous matrix of the invention batch between become
Different coefficient is not more than 5.45%, and the interassay coefficient of variation of cell opposite proliferation rate is not more than 5.74%, tensile strength batch between become
Different coefficient is not more than 8.75%, is substantially reduced compared with comparative example 1-3, illustrates Acellular cartilaginous matrix quality of the invention more
Reliable and stable, homogeneity significantly improves.Moreover, compared with embodiment 4-6, Acellular cartilaginous matrix obtained in embodiment 1-3
DNA residual quantity, tensile strength variation lines have significant decrease, cell opposite proliferation rate and tensile strength are obviously improved, table
It is bright by the way that hypotonic, trypsin treatment condition, the optimization of nuclease concentration and thimerosal is conducive to further increase obtained
Acellular cartilaginous matrix quality stability.
Experimental example 4
By the natural cartilage group in Acellular cartilaginous matrix made from embodiment 1 and embodiment 1 without taking off cell processing
Progress hematoxylin eosin staining (HE dyeing) respectively is knitted, specific experimental method is as follows: taking off cellular cartilage made from Example 1
Matrix and the cartilaginous tissue without taking off cell processing, respectively experimental group and control group, the paraformaldehyde that each group is all made of 4% are solid
It is fixed for 24 hours, gradient alcohol dehydration, dimethylbenzene is transparent, waxdip, packet, slice, and slice thickness is 5 μm, obtains paraffin section.Then will
Paraffin section dewaxes each 10min in dimethylbenzene, successively enters dehydrated alcohol, 95% ethyl alcohol, each 5min of 85% ethyl alcohol, tap water punching
Wash 1-2min.Then 3-5min is contaminated with haematoxylin dye liquor, tap water rinses 1-2min.1% hydrochloride alcohol breaks up the several seconds.1% ammonia
Aqueous solution returns the blue several seconds, then washes 1-2min.Enter eosin stain dye 1-3min.Then dehydrated alcohol is dehydrated each 1min twice,
Transparent several minutes of dimethylbenzene.Neutral gum mounting, microscopy, as a result as shown in Figure 1.
The results show that the cell-free ingredient of Acellular cartilaginous matrix made from embodiment 1 exists, cell removing is clean, cell
The structure of epimatrix is consistent with natural cartilage tissue, illustrates that the extra-cellular matrix structure of Acellular cartilaginous matrix obtained has been kept
It is whole.
Experimental example 5
It is handled without de- cell using DAPI stain in Acellular cartilaginous matrix made from embodiment 1 and embodiment 1
Cartilaginous tissue in DNA dyed, specific experimental method is as follows: Acellular cartilaginous matrix made from Example 1 and not
The cartilaginous tissue of cell processing, respectively experimental group and control group are taken off, the paraformaldehyde that each group is all made of 4% is fixed for 24 hours, ladder
Dehydration of alcohol is spent, dimethylbenzene is transparent, waxdip, packet, slice, and slice thickness is 5 μm, obtains paraffin section.By paraffin section in two
It dewaxes in toluene, successively enters dehydrated alcohol, 95% ethyl alcohol, each 5min of 85% ethyl alcohol, tap water flushing 1-2min.DAPI is added to work
Liquid, room temperature, which is protected from light, is incubated for 10min, and PBS is rinsed three times, each 5min.Fluorescence microscopy microscopic observation after mounting, as a result such as Fig. 2 institute
Show.
The results show that there is stronger fluorescence in natural cartilaginous tissue, illustrate have without the cartilaginous tissue for taking off cell processing
Nuclear fraction, and unstressed configuration in Acellular cartilaginous matrix illustrate the cell-free nuclear composition of Acellular cartilaginous matrix obtained.
Experimental example 6
To in Acellular cartilaginous matrix made from embodiment 1 and embodiment 1 without take off cell processing cartilaginous tissue into
Row antigen ɑ-gal detection, specific experimental method is as follows: Acellular cartilaginous matrix made from Example 1 and without de- cell at
The cartilaginous tissue of reason, respectively experimental group and control group, the paraformaldehyde that each group is all made of 4% is fixed for 24 hours, and graded ethanol is de-
Water, dimethylbenzene is transparent, waxdip, packet, slice, and slice thickness is 5 μm.
Slice is placed in 65 DEG C of ovens, 1-2h is baked.Then slice is dewaxed in dimethylbenzene, successively enters dehydrated alcohol,
95% ethyl alcohol, each 1min of 85% ethyl alcohol, each 1min of 80% ethyl alcohol, 70% ethyl alcohol each 1min, PBS rinse 3min, and 3 times.Then it sets
In pre-configured sodium citrate antigen retrieval buffers, in Pressure method pot, working solution is added and is heated to boiling, is put into and cuts
Piece continues to be heated to pressure limiting valve jet rotation beginning timing 2.5min after covering tightly.Leave heat source, natural cooling 5min, tap water
Rinse, after being cooled to room temperature take out slice, PBS rinse 3min, 3 times.With 3% dioxygen water seal 10 minutes, PBS rinsed 3min, and 3
It is secondary, the PBST around tissue is dried, closes 1h with Zhong Shan Golden Bridge normal sheep serum, incline serum deprivation, does not wash.Primary antibody is added, overnight
(ɑ-gal antibody carries fluorescence, article No. (I21412) 1:2000).PBST, 3 minutes, 3 times, mounting, fluorescence microscopy was under the microscope
Slice, as a result as shown in Figure 3.
The results show that having fluorescence without taking off in the cartilaginous tissue that cell is handled, illustrate there is ɑ-gal antigen presentation, and it is de- thin
There is no fluorescence in Acellular cartilaginous matrix after born of the same parents, illustrates no ɑ-gal antigen presentation.
Experimental example 7
To in Acellular cartilaginous matrix made from embodiment 1 and embodiment 1 without take off cell processing cartilaginous tissue into
Row improvement tri- color of Masson dye, specific experimental method is as follows: Acellular cartilaginous matrix made from Example 1 and without take off cell
The cartilaginous tissue of processing, respectively experimental group and control group, the paraformaldehyde that each group is all made of 4% is fixed for 24 hours, and graded ethanol is de-
Water, dimethylbenzene is transparent, waxdip, packet, slice, and slice thickness is 5 μm, obtains paraffin section.
Paraffin section is immersed into bouin fixer, 2h mordant dyeing in 37 degree of incubators, the yellow that flowing water is rinsed to slice disappears
It loses；Celestine blue 2-3min, is slightly washed；Mayer haematoxylin 8min, is slightly washed；Acidic ethanol breaks up liquid and breaks up the several seconds；Flowing water punching
Wash (anti-blue)；Ponceaux magenta 8min, is slightly washed；Phosphomolybdic acid impregnates；Incline upper liquid, slice is not washed with water, directly instillation aniline
Blue dye liquor impregnates；Weak acid solution handles 2min；95% ethyl alcohol fast dewatering, dehydrated alcohol are dehydrated twice, each 5-10s；Transparent,
Mounting.Fluorescence microscopy microscopic observation after mounting, as a result as shown in Figure 4.
The results show that explanation has nuclear fraction and fibre structure without taking off the cartilaginous tissue of cell processing, and pass through de- thin
Born of the same parents handle Acellular cartilaginous matrix obtained, and fibre structure saves complete, cell-free nuclear composition.
Experimental example 8
To in Acellular cartilaginous matrix made from embodiment 1 and embodiment 1 without take off cell processing cartilaginous tissue into
The detection of row II Collagen Type VI immunohistochemistry, specific experimental method is as follows: Acellular cartilaginous matrix made from Example 1 and without de-
The cartilaginous tissue of cell processing, respectively experimental group and control group, the paraformaldehyde that each group is all made of 4% is fixed for 24 hours, gradient wine
Essence dehydration, dimethylbenzene is transparent, waxdip, packet, slice, and slice thickness is 5 μm.
Slice is placed in 65 DEG C of ovens, 1-2h is baked.Then slice is dewaxed in dimethylbenzene, successively enters dehydrated alcohol,
95% ethyl alcohol, each 1min of 85% ethyl alcohol, each 1min of 80% ethyl alcohol, 70% ethyl alcohol each 1min, PBS rinse 3min, and 3 times.Then it sets
In pre-configured sodium citrate antigen retrieval buffers, in Pressure method pot, working solution is added and is heated to boiling, is put into and cuts
Piece continues to be heated to pressure limiting valve jet rotation beginning timing 2.5min after covering tightly.Leave heat source, natural cooling 5min, tap water
Rinse, after being cooled to room temperature take out slice, PBS rinse 3min, 3 times.With 3% dioxygen water seal 10 minutes, PBS rinsed 3min, and 3
It is secondary, the PBST around tissue is dried, closes 1h with Zhong Shan Golden Bridge normal sheep serum, incline serum deprivation, does not wash.Primary antibody is added, overnight
(Collagen II antibody, article No. (ab34712), 1:500).PBST, 3 minutes, 3 times, mounting, fluorescence microscopy was cut under the microscope
Piece, as a result as shown in Figure 5.
The results show that explanation has nuclear fraction and II Collagen Type VI without the cartilaginous tissue for taking off cell processing, through taking off cell
Acellular cartilaginous matrix obtained is handled, II Collagen Type VI saves complete, cell-free nuclear composition.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of preparation method of Acellular cartilaginous matrix, which is characterized in that cartilaginous tissue is passed sequentially through Hypotonic treatment, pancreas egg
Cartilage matrix is made in white enzymatic treatment, detergent-treatment and nucleic acid enzymatic treatment.
2. preparation method according to claim 1, which is characterized in that in Hypotonic treatment, cartilaginous tissue is added hypotonic
It is incubated in solution, is incubated for 15-30h at room temperature；
Preferably, the Tris-Hcl solution that the hypotonic solution is 10mM and pH is 7-8.
3. preparation method according to claim 1 or 2, which is characterized in that in trypsin treatment, by cartilaginous tissue plus
Enter in trypsin solution, the heating water bath 6-24h at 37 DEG C；
Preferably, the trypsin solution is the PBS solution containing 0.10-0.50% (m/v) trypsase.
4. preparation method according to claim 1 to 3, which is characterized in that in detergent-treatment, by cartilage group
It knits and is added in detergent solution, oscillation treatment 15-30h at being 15-25 DEG C in temperature；
Preferably, the detergent solution is the Tris-Hcl solution of the 8-10mM containing 0.05-0.3%SDS.
5. preparation method according to any one of claims 1-4, which is characterized in that in nucleic acid enzymatic treatment, by cartilage group
It knits and is added in buffered cell liquid, the DNA enzymatic of 30-60U/ml and the RNA enzyme of 0.5-4U/ml, the water-bath at 35-40 DEG C is then added
Heat 6-20h；
Preferably, the buffered cell liquid be the magnesium chloride containing 5-30mM Tris-Hcl solution, the Tris-Hcl solution it is dense
Degree is 30-60mM, pH 7-8.
6. any preparation method in -5 according to claim 1, which is characterized in that before Hypotonic treatment further include to soft
Bone tissue carries out multigelation processing；
Preferably, in multigelation processing, cartilaginous tissue is placed at -70~-80 DEG C and freezes 2-4h, is subsequently placed in 15-25
Thaw 1-6h at DEG C, multigelation 1-6 times.
7. any preparation method in -6 according to claim 1, which is characterized in that in front of nucleic acid enzymatic treatment further including
Property Protease Treatment；
Preferably, in neutral protein enzymatic treatment, cartilaginous tissue is added to the physiology salt of the neutral proteinase containing 300-700U/L
In water, the heating water bath 4-12h at 30-40 DEG C；
It preferably, further include α -1,3- galactoside enzyme solutions processing, in α -1,3- galactolipin after neutral protein enzymatic treatment
In glycosides enzymatic treatment, cartilaginous tissue is added to α -1 of the g/mL of μ containing 10-40, in the PBS solution of 3- galactosidase, at 25-37 DEG C
Under, it impregnates 1-6 hours.
8. any preparation method in -7 according to claim 1, which is characterized in that after nucleic acid enzymatic treatment further include going out
Bacterium processing；
Preferably, in sterilization treatment, cartilaginous tissue is added in thimerosal and impregnates 1-6h, the thimerosal is to contain 0.05-
The PBS solution of 0.4vt% acetic acid and 0.05-0.4wt% sodium hypochlorite, or
PBS solution containing 0.05-0.4vt% acetic acid and 0.05-0.4vt%84 thimerosal.
9. preparation method described in -8 according to claim 1, which is characterized in that before cartilaginous tissue further includes before Hypotonic treatment
Cleaning treatment；It further include at intermediate cleaning after Hypotonic treatment, trypsin treatment, detergent-treatment and nucleic acid enzymatic treatment
Reason；Cleaning treatment after further including after nucleic acid enzymatic treatment；
Preferably, in preceding cleaning treatment, cartilaginous tissue is added in physiological saline and is impregnated, it is clear that ultrasound in ultrapure water is then added
It washes, then with ultrapure water continual rinsing；
Preferably, in intermediate cleaning treatment, cartilaginous tissue is added in ultrapure water and is cleaned by ultrasonic, then is persistently rushed with ultrapure water
It washes；
Preferably, in rear cleaning treatment, cartilaginous tissue is added in sterile PBS and is impregnated.
10. Acellular cartilaginous matrix made from any preparation method in a kind of claim 1-9.
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| CN114796615A (en) * | 2022-04-20 | 2022-07-29 | 诺一迈尔（苏州）医学科技有限公司 | Cartilage acellular matrix and preparation method thereof |
| CN114796615B (en) * | 2022-04-20 | 2023-08-25 | 诺一迈尔（苏州）医学科技有限公司 | Cartilage acellular matrix and preparation method thereof |
| CN115444987A (en) * | 2022-08-18 | 2022-12-09 | 江西中洪博元生物技术有限公司 | Cartilage tissue acellular hydrogel scaffold and preparation method and application thereof |
| CN115444987B (en) * | 2022-08-18 | 2023-07-11 | 江西中洪博元生物技术有限公司 | Cartilage tissue decellularized hydrogel scaffold and preparation method and application thereof |
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Application publication date: 20190416 |