CN106075584A - Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof - Google Patents
Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof Download PDFInfo
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- CN106075584A CN106075584A CN201610710756.4A CN201610710756A CN106075584A CN 106075584 A CN106075584 A CN 106075584A CN 201610710756 A CN201610710756 A CN 201610710756A CN 106075584 A CN106075584 A CN 106075584A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—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
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- 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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Abstract
The invention discloses a kind of syringeability cartilage and take off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof.Of the same race or xenogenesis cartilage are prepared cartilage cell epimatrix through de-cell process by the present invention, of the same race or bone-xenograft tissue are prepared decalcified bone matrix through defat, decalcification, de-cell process simultaneously, again through pepsin digestion after the two mixing according to a certain percentage, under neutral pH, 37 DEG C and suitable salt concentration conditions, it is solidified into syringeability hydrogel.The cartilage using the method for the invention to prepare takes off extracellular matrix mixing decalcified bone matrix hydrogel and has good biodegradability, it is most important that have syringeability；Processing simultaneously as carry out de-cell, immunogenicity is substantially reduced, and has preferable biocompatibility, and biocompatible reaction reduces；The present invention draws materials simply, easy to operate, can be used for various cartilage defect and fills and Wicresoft's orthopaedics repair of cartilage treatment, has good potential applicability in clinical practice.
Description
Technical field
The present invention relates to bioengineered tissue technology, specifically a kind of syringeability cartilage takes off extracellular matrix mixing decalcification
Bone matrix hydrogel and preparation method thereof.
Background technology
Articular cartilage damage, defect is caused to be always orthopaedics by factors such as regression, wound, various inflammation, tumors clinically
Clinical problems faced, the articular cartilage overwhelming majority belongs to hyaline cartilage, depletion of blood confession, lymphoid tissue and innervation, self-regeneration
Limited in one's ability, self-regeneration ability.Articular cartilage damage treatment becomes one of current osteoarthrosis surgery difficult problem.
Millions of patient is had to accept treatment because of articular cartilage damage clinically every year.Native cartilage is transplanted and allosome at present
Cartilage transplantation is respectively provided with certain limitation, and native cartilage is transplanted and produced new cartilage defect, and homogenous cartilage is transplanted and be there is immunity
Originality is reacted.Since twentieth century eighties, chondrocyte treatment articular cartilage damage causes people's interest, i.e. soft
Transplant chondrocyte at Cranial defect and realize the reparation of cartilage defect.But transplant merely chondrocyte, cell cannot plant and
Propagation, causes poor effect.So for a long time, a difficult problem for Orthopedic Clinical is always perplexed in the regeneration of osteocartilaginous and subchondralo bone injury and reparation
One of, also it is one of clinical focus with basic research.In recent years, along with macromolecule medical material, biochemistry, materialogy,
The multi-disciplinary fast development of molecular biology, preclinical medicine, clinical medicine, cartilage tissue engineered have also been obtained rapidly development,
New approach is provided for solving this difficult problem.
For these reasons, people are to find a kind of preferably cartilage repair material antigenicity low and have cartilage and lure
Lead and cartilage characteristic.The reparation cartilage technology that home and overseas is generally acknowledged at present is that (Autologous Chondrocyte moves second filial generation ACI technology
Plant): MACI technology (transplanting of autologous cartilage compound cells epimatrix), have been used to clinic at present, concrete grammar is exactly can be internal
The timbering material of degraded is combined chondrocyte and is then implanted to cartilage defect.
The late 20th century starts the research of de-cell tissue extracellular matrix, and cell is considered as immunogenic root,
The cell sloughing tissue just removes most of immunizing antigen composition of tissue.Tissue can remove cell through de-cell step
Film, cytoplasmic components and intracellular soluble protein etc. can induce the material of immunologic rejection, and remaining extracellular matrix forms
There is certain biological activity.The acellular matrix of the most a lot of tissues is as the timbering material in organizational project, such as myocardium
Extracellular matrix injection for curing myocardial infarction is applied to clinic.
Surgical clinical operation technique presents Wicresoft's trend.Bone surgery is also such, especially Wicresoft's joint surgery development
Rapidly, it is to avoid conventional cutting operation, cartilage injury Wicresoft prothesis gets more and more for syringeability bone-grafting material demand.
Hydrogel is the polymer with hydrophilic tridimensional network, cannot dissolve in water, have mobility and
Good biocompatibility, has a syringeability, and gelatinizing-in-situ is simple to operate.
Osteochondral tissue is made up of chondrocyte and extracellular matrix, and extracellular matrix is many by II Collagen Type VI and aminoglucose
Polysaccharide (GAG) forms.Articular cartilage about 70% moisture, 20% II Collagen Type VI, 5% proteoglycan and 5% chondrocyte.Cartilage is thin
Born of the same parents are positioned in the loculus cartilage lacuna of cartilage matrix.Cartilage capsule is positioned at around lacuna, rich in chondroitin sulfate.Cartilage is thin
Born of the same parents, by Derived from Mesenchymal Stem Cells, are positioned in extracellular matrix.Articular chondrocytes epimatrix main component is proteoglycan, right
Cell and form thereof have support and regulation effect, regulation cellular metabolism, the signal conduction of differentiation and proliferation equimolecular level.Joint
In cartilage, collagen is based on typeⅡ Collagen, accounts for the 90%～95% of chondrigen total amount.
Osseous tissue is hard connective tissue, is made up of cell and substrate.Substrate is divided into organic matter and inanimate matter, organic
Containing substantial amounts of collagen fiber, inanimate matter is substantial amounts of inorganic calcium salt, and collagen is embedded in calcium salt substrate.
Collagen is human body and vertebrate major structural protein, is supporting tissue and connective tissue (skin, flesh key and bone
The organic moiety of bone) main component, tropocollagen molecule structure is three polypeptide chains, in three-dimensional spiral structure, has immunologic inertia,
It is suitable as cartilage tissue engineering rack, dissimilar cell can be promoted to bond, grow, break up and mobile.Collagen is a kind of sky
Right hydrogel, can meet the clinical needs to injectivity bone-grafting material.
Summary of the invention
The present invention is contemplated to provide a kind of new injectivity bone-grafting material met clinical needs, and the one proposed can
Injectivity cartilage takes off extracellular matrix mixing decalcified bone matrix (DBM) hydrogel and preparation method thereof.
The present invention realizes according to techniques below scheme.
A kind of syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel, described hydrogel be by of the same race or
Xenogenesis cartilage processes through de-cell, and of the same race or bone-xenograft tissue processes through defat, decalcification, de-cell, and the two is according to necessarily
Formed after ratio mixing dissolves under weak acid, at physiological temp, neutral pH and suitable salinity (0.O1～0.1mol/
L) it is solidified under conditions of and there is certain viscosity and the ossein hydrogel containing the osteogenic activity factor.
Described of the same race or xenogenesis cartilage and osseous tissue are respectively derived from pig or cattle or sheep or the cartilage of people and spongy bone.
Described hydrogel contains cartilage cell epimatrix and spongy bone extracellular matrix, mucopolysaccharide, type i collagen, II type glue
Former.
A kind of above-mentioned syringeability cartilage takes off the preparation method of extracellular matrix mixing decalcified bone matrix hydrogel, bag
Include following steps:
A prepares cartilage and takes off extracellular matrix lyophilizing sponge
1. soak of the same race or xenogenesis cartilage block with the Tris-HCl buffer containing Phenylmethanesulfonyl fluoride, and cartilage is pulverized, distillation
Water rinses repeatedly；
2. the Tris-HCl buffer concussion 12～24h containing Triton X-100 and Phenylmethanesulfonyl fluoride is added；
3. ultra-pure water repeatedly rinses, drains；
4. adding DNase I and RNase A concussion digestion 12～24h, ultra-pure water rinses repeatedly；
5. lyophilization；
B prepares the lyophilizing sponge Han decalcified bone matrix
1. soak of the same race or bone-xenograft tissue with the Tris-HCl buffer containing Phenylmethanesulfonyl fluoride, and osseous tissue is pulverized, steam
Distilled water is rinsed repeatedly；
2. carrying out defat 12～72h with chloroform and methanol, distilled water rinses repeatedly；
3. carrying out decalcification 12～72h with HCl solution, distilled water rinses repeatedly；
4. CaCl is added2Solution stirring 1～3h, distilled water rinses repeatedly；Adding EDTA solution stirring 1～3h, distilled water is repeatedly
Rinse；Adding LiCl solution stirring 1～3h, distilled water rinses repeatedly；
5. the Tris-HCl buffer concussion 12～24h containing Triton X-100 and Phenylmethanesulfonyl fluoride is added；
6. ultra-pure water repeatedly rinses, drains；
7. adding DNase I and RNase A concussion digestion 12～24h, ultra-pure water rinses repeatedly；
8. lyophilization；
The c cartilage cell epimatrix mixing preparation containing decalcified bone matrix hydrogel
The lyophilizing cartilage cell epimatrix obtained in blend step a and step b the most according to a certain percentage and freeze drying bone substrate, use
HCl solution dissolves and adds pepsin, stirs 12～96h；
2. pH value of solution is adjusted to neutrality, adds 10 × PBS and 1 × PBS mixing；
3. place under the conditions of 20～37 DEG C and be solidified into hydrogel in 20～60 minutes.
Described step a 1. with b 1. in cartilage and osseous tissue be ground into 0～5mm × 0～5mm × 0～5mm size
Grain.
Described step a the most 2. with b the most 5. in Tris-HCl buffer concentration be 0.01～0.1mol/L, pH7.4～7.5；
Step a the most 2. with b the most 5. in Phenylmethanesulfonyl fluoride concentration be 0.035～0.35 mmol/L；Step a 2. with b 5. in Triton
X-100 concentration is 0.1～1 mmol/L；The volume ratio of step b 2. middle chloroform and methanol is 1:1～2:1；3. middle HCl is dense for step b
Degree 0.01～0.5 mol/L；Step b 4. middle CaCl2The concentration of solution is 2～3.6mol/L, and the concentration of EDTA solution is 0.01
～the concentration of 0.5mol/L, LiCl solution is 2～8mol/L；4. step a is respectively with RNase A concentration with b 7. middle DNase I
10～50 U/ml, 1～5 U/ml.
Described step c 1. middle HCl concentration is 0.01～0.5 mol/L；Pepsin: cartilage cell epimatrix is freeze-dried mixed
Bone matrix=50～150mg:1g；The step c 2. middle pH value that adjusts is to neutral NaOH concentration 0.01～1mol/L.
The mass ratio of described step c 1. middle cartilage cell epimatrix and freeze drying bone substrate is less than 1:1.
The mass ratio of described step c 1. middle cartilage cell epimatrix and freeze drying bone substrate is 1:2.
Described step a, b are all carried out in addition to digestion under the conditions of 0-4 DEG C；2. step c is carried out under the conditions of 0～4 DEG C.
Present invention obtains following beneficial effect.
In the present invention, the II Collagen Type VI in the cartilage extracellular matrix obtained after cartilage takes off cell can fill between inducing bone marrow
Matter stem cell (BMSCs) differentiating cartilage-forming cell also has the feature of chondrocyte, and II Collagen Type VI can be 37 DEG C of conditions
Lower formation hydrogel, has syringeability, good biodegradability and biocompatibility.Spongy bone through decalcification, defat,
De-cells etc. process, and eliminate most noncollagen protein in addition to self-bone grafting forms albumen (BMP), greatly reduce bone and repair
The antigenicity of multiple material, and there is good histocompatibility and degradability.Its hydrogel state has natural grid three
Dimension pore structure, has certain elasticity, and syringeability is strong, and forms albumen containing self-bone grafting, can support and Induction of chondrocytes
Plantation and propagation.Water is made by compound for cell epimatrix material natural to cartilage extracellular matrix and spongy bone extracellular matrix both
Gel, can be as the support of chondrocyte compound in soft tissue engineering, and two kinds of extracellular matrix materials are had complementary advantages, and can make up
The most not enough.Spongy bone extracellular matrix contains BMP-2, and it has facilitation, aminoglucose to the differentiation of chondrocyte
Polysaccharide (GAG) and II Collagen Type VI are that chondrocyte secretion produces, and BMP-2 can improve its secretion level.The present invention takes
Material is simple, easy to operate, low cost, can be used for cartilage defect and fills and Wicresoft's orthopaedics repair of cartilage treatment, has good facing
Bed application prospect.
Accompanying drawing explanation
Fig. 1 is the front view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel；
Fig. 2 is the side view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel；
Fig. 3 is the front view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel lyophilizing sponge；
Fig. 4 is the top view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel lyophilizing sponge；
Fig. 5 is that syringeability cartilage of the present invention takes off under extracellular matrix mixing decalcified bone matrix hydrogel lyophilizing substantially microscope mirror
Figure；
Fig. 6 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel scanning electron microscope (SEM) photograph (× 500)；
Fig. 7 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel HE colored graph (× 100)；
Fig. 8 be syringeability cartilage of the present invention take off extracellular matrix mixing decalcified bone matrix hydrogel sarranine " O " colored graph (×
100)；
Fig. 9 be syringeability cartilage of the present invention take off extracellular matrix mixing decalcified bone matrix hydrogel Toluidine blue staining figure (×
100)；
Figure 10 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel type i collagen SABC
Figure；
Figure 11 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel II Collagen Type VI SABC
Figure.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment the present invention is described further.
A kind of syringeability cartilage of the present invention takes off the preparation method of extracellular matrix mixing decalcified bone matrix hydrogel such as
Under:
A prepares cartilage and takes off extracellular matrix lyophilizing sponge (following operation in addition to digestion all under the conditions of 0～4 DEG C)
Taking fresh pig hyaline cartilage, 1. with pH7.4, the Tris-HCl buffer of concentration 0.01mol/L is (containing 0.035 mmol/L
Phenylmethanesulfonyl fluoride) soak hyaline cartilage block, with pulverizer (BILON, model DS-1), cartilage made 5mm × 5mm × 5mm big
Fine granularity, distilled water rinses repeatedly；
2. the Tris-HCl buffer shake containing 1 mmol/L Triton X-100 and 0.035 mmol/L Phenylmethanesulfonyl fluoride is added
Swing 12～24h；
3. ultra-pure water repeatedly rinses, drains；
4. adding 50U/ml DNase I and 5U/mlRNase A and shake digestion 12h at 37 DEG C, ultra-pure water rinses repeatedly；
5. lyophilization ,-20 DEG C save backup.
B preparation is containing decalcified bone matrix lyophilizing sponge (following operation in addition to digestion all under the conditions of 0～4 DEG C)
Take fresh pig spongy bone, 1. with the Tris-HCl buffer of pH7.4,0.01mol/L (containing 0.035 mmol/L benzyl sulphur
Acyl fluorides) soak spongiosa bone piece, with pulverizer (BILON, model DS-1), spongy bone made 5mm × 5mm × 5mm size particles
Shape, distilled water rinses repeatedly；
2. with chloroform: methanol=1:1 mixed liquor defat 12～72h, distilled water rinses repeatedly；
3. with 0.01mol/L HCl solution (25～30ml/g bone) decalcification 12～72h, distilled water rinses repeatedly；
4. 2mol/L CaCl is used2Stirring 1h, distilled water rinses repeatedly；0.5mol/L EDTA stirs 1h, and distilled water rinses repeatedly；
8mol/L LiCl stirs 1h, and distilled water rinses repeatedly；
5. the Tris-HCl buffer shake containing 1 mmol/L Triton X-100 and 0.035 mmol/L Phenylmethanesulfonyl fluoride is added
Swing 12～24h；
6. ultra-pure water repeatedly rinses, drains；
7. adding 50U/ml DNase I and 5U/ml RNase A and shake digestion 12h at 37 DEG C, ultra-pure water rinses repeatedly；
8. lyophilization ,-20 DEG C save backup；
The c cartilage cell epimatrix mixing preparation containing decalcified bone matrix hydrogel
1. take 0.5g cartilage cell epimatrix mixing 1g lyophilizing sponge Han decalcified bone matrix, be dissolved in 20ml 0.01mol/L HCl molten
Liquid, adds pepsin 150mg, stirs 24h；
2. under the conditions of 4 DEG C, take 0.5ml cell mixing epimatrix, adjust pH value to neutral with 0.1mol/L NaOH, add
0.11ml 10 × PBS and 0.29ml 1 × PBS mixing；
3. place 20 minutes at 37 DEG C, hydrogel can be solidified into.
Fig. 1,2 it is to use the cartilage prepared of the method for the invention to take off extracellular matrix mixing decalcified bone matrix hydrogel,
Hydrogel diameter 1cm, highly 1cm, milky, smooth surface moistens.
Fig. 3,4 it is to use the cartilage prepared of the method for the invention to take off extracellular matrix mixing decalcified bone matrix hydrogel
Dried frozen aquatic products, outward appearance off-white color, flexible, similar sponge.
Fig. 5 is that the cartilage using the method for the invention to prepare takes off freezing of extracellular matrix mixing decalcified bone matrix hydrogel
Seeing under dry product substantially microscope, pore-creating character is good, similar sponge.
The cartilage that the display of Fig. 6 scanning electron microscope (SEM) photograph uses the method for the invention to prepare takes off extracellular matrix mixing decalcification bone base
Matter hydrogel is rich in space, and hole intercommunication.
Fig. 7 show use the cartilage prepared of the method for the invention take off extracellular matrix mixing decalcified bone matrix hydrogel Lignum Sappan with
Yihong (HE) dyeing has no that (confirming that de-cell is thorough, cell is immunogenic main source to Radix Indigoferae Carlesii dye nucleus, such water
The rejection of gel substantially reduces), the extracellular matrix of the most visible eosin stains.
Fig. 8 shows that the cartilage using the method for the invention to prepare takes off extracellular matrix mixing decalcified bone matrix hydrogel kind
Red " O " dyes: the proteoglycan (GAG) of visible stain.
Fig. 9 shows that the cartilage using the method for the invention to prepare takes off extracellular matrix mixing decalcified bone matrix hydrogel first
Aniline blue dyeing display staining for collagen is positive.
Figure 10 shows that the cartilage using the method for the invention to prepare takes off extracellular matrix mixing decalcified bone matrix hydrogel
SABC NTx is positive.
Figure 11 shows that the cartilage using the method for the invention to prepare takes off extracellular matrix mixing decalcified bone matrix hydrogel
SABC II Collagen Type VI is positive.
Claims (10)
1. a syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel, it is characterised in that: described hydrogel
Being to be processed through de-cell by of the same race or xenogenesis cartilage, of the same race or bone-xenograft tissue processes through defat, decalcification, de-cell, and two
What person was formed after mixing according to a certain percentage dissolves under weak acid, at physiological temp, neutral pH and the bar of suitable salinity
It is solidified under part and there is certain viscosity and the ossein hydrogel containing the osteogenic activity factor.
A kind of syringeability cartilage the most according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel, its
It is characterised by: described of the same race or xenogenesis cartilage and osseous tissue are respectively derived from pig or cattle or sheep or the cartilage of people and spongy bone.
A kind of syringeability cartilage the most according to claim 2 takes off extracellular matrix mixing decalcified bone matrix hydrogel, its
It is characterised by: described hydrogel contains cartilage cell epimatrix and spongy bone extracellular matrix, mucopolysaccharide, type i collagen, II type glue
Former.
4. the syringeability cartilage described in a claim 1 takes off the preparation side of extracellular matrix mixing decalcified bone matrix hydrogel
Method, it is characterised in that: comprise the following steps:
A prepares cartilage and takes off extracellular matrix lyophilizing sponge
1. soak of the same race or xenogenesis cartilage block with the Tris-HCl buffer containing Phenylmethanesulfonyl fluoride, and cartilage is pulverized, distillation
Water rinses repeatedly；
2. the Tris-HCl buffer concussion 12～24h containing Triton X-100 and Phenylmethanesulfonyl fluoride is added；
3. ultra-pure water repeatedly rinses, drains；
4. adding DNase I and RNase A concussion digestion 12～24h, ultra-pure water rinses repeatedly；
5. lyophilization；
B prepares the lyophilizing sponge Han decalcified bone matrix
1. soak of the same race or bone-xenograft tissue with the Tris-HCl buffer containing Phenylmethanesulfonyl fluoride, and osseous tissue is pulverized, steam
Distilled water is rinsed repeatedly；
2. carrying out defat 12～72h with chloroform and methanol, distilled water rinses repeatedly；
3. carrying out decalcification 12～72h with HCl solution, distilled water rinses repeatedly；
4. CaCl is added2Solution stirring 1～3h, distilled water rinses repeatedly；Adding EDTA solution stirring 1～3h, distilled water rushes repeatedly
Wash；Adding LiCl solution stirring 1～3h, distilled water rinses repeatedly；
5. the Tris-HCl buffer concussion 12～24h containing Triton X-100 and Phenylmethanesulfonyl fluoride is added；
6. ultra-pure water repeatedly rinses, drains；
7. adding DNase I and RNase A concussion digestion 12～24h, ultra-pure water rinses repeatedly；
8. lyophilization；
The c cartilage cell epimatrix mixing preparation containing decalcified bone matrix hydrogel
The lyophilizing cartilage cell epimatrix obtained in blend step a and step b the most according to a certain percentage and freeze drying bone substrate, use
HCl solution dissolves and adds pepsin, stirs 12～96h；
2. pH value of solution is adjusted to neutrality, adds 10 × PBS and 1 × PBS mixing；
3. place under the conditions of 20～37 DEG C and be solidified into hydrogel in 20～60 minutes.
A kind of syringeability cartilage the most according to claim 4 takes off the system of extracellular matrix mixing decalcified bone matrix hydrogel
Preparation Method, it is characterised in that: described step a 1. with b 1. in cartilage and osseous tissue be ground into 0～5mm × 0～5mm × 0～
The granule of 5mm size.
A kind of syringeability cartilage the most according to claim 4 takes off the system of extracellular matrix mixing decalcified bone matrix hydrogel
Preparation Method, it is characterised in that: described step a the most 2. with b the most 5. in Tris-HCl buffer concentration be 0.01～0.1mol/L,
PH7.4～7.5；Step a the most 2. with b the most 5. in Phenylmethanesulfonyl fluoride concentration be 0.035～0.35 mmol/L；2. and b step a
Middle Triton X-100 concentration is 0.1～1 mmol/L；The volume ratio of step b 2. middle chloroform and methanol is 1:1～2:1；Step
Rapid b 3. middle HCl concentration 0.01～0.5 mol/L；Step b 4. middle CaCl2The concentration of solution is 2～3.6mol/L, EDTA solution
Concentration be 0.01～0.5mol/L, the concentration of LiCl solution is 2～8mol/L；Step a 4. with b 7. in DNase I with
RNase A concentration is respectively 10～50 U/ml, 1～5 U/ml.
A kind of syringeability cartilage the most according to claim 4 takes off the system of extracellular matrix mixing decalcified bone matrix hydrogel
Preparation Method, it is characterised in that: described step c 1. middle HCl concentration is 0.01～0.5 mol/L；Pepsin: the outer base of chondrocyte
Freeze-dried mixed bone matrix=50 of matter～150mg:1g；The step c 2. middle pH value that adjusts is to neutral NaOH concentration 0.01～1mol/L.
A kind of syringeability cartilage the most according to claim 4 takes off the system of extracellular matrix mixing decalcified bone matrix hydrogel
Preparation Method, it is characterised in that: the mass ratio of described step c 1. middle cartilage cell epimatrix and freeze drying bone substrate is less than 1:1.
A kind of syringeability cartilage the most according to claim 8 takes off the system of extracellular matrix mixing decalcified bone matrix hydrogel
Preparation Method, it is characterised in that: the mass ratio of described step c 1. middle cartilage cell epimatrix and freeze drying bone substrate is 1:2.
A kind of syringeability cartilage the most according to claim 4 takes off extracellular matrix mixing decalcified bone matrix hydrogel
Preparation method, it is characterised in that: described step a, b are all carried out in addition to digestion under the conditions of 0～4 DEG C；Step c is 2. at 0～4 DEG C of bar
Carry out under part.
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