CN106434538A - Three-dimensional chondrocyte culture method, made chondrocyte and application - Google Patents
Three-dimensional chondrocyte culture method, made chondrocyte and application Download PDFInfo
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- 238000012136 culture method Methods 0.000 title claims abstract description 21
- 210000001612 chondrocyte Anatomy 0.000 title abstract description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 20
- 210000000845 cartilage Anatomy 0.000 claims abstract description 13
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 11
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 11
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 4
- 210000003321 cartilage cell Anatomy 0.000 claims description 28
- 210000001519 tissue Anatomy 0.000 claims description 28
- 239000003462 bioceramic Substances 0.000 claims description 13
- 210000000130 stem cell Anatomy 0.000 claims description 12
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 9
- 206010007710 Cartilage injury Diseases 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000010146 3D printing Methods 0.000 claims description 5
- 239000001569 carbon dioxide Substances 0.000 claims description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000001506 calcium phosphate Substances 0.000 claims description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 2
- 235000011010 calcium phosphates Nutrition 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 9
- 206010060820 Joint injury Diseases 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 5
- 208000027418 Wounds and injury Diseases 0.000 abstract description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 abstract description 3
- 208000014674 injury Diseases 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 2
- 230000035755 proliferation Effects 0.000 abstract description 2
- 102000000503 Collagen Type II Human genes 0.000 abstract 1
- 108010041390 Collagen Type II Proteins 0.000 abstract 1
- 229910010293 ceramic material Inorganic materials 0.000 abstract 1
- 102000002734 Collagen Type VI Human genes 0.000 description 6
- 108010043741 Collagen Type VI Proteins 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010057178 Osteoarthropathies Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010061762 Chondropathy Diseases 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 210000000579 abdominal fat Anatomy 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000009288 screen filtration Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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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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- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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Abstract
The invention belongs to the technical field of cells and particularly relates to a three-dimensional chondrocyte culture method, a made chondrocyte and application. The three-dimensional chondrocyte culture method comprises the steps that a chondrocyte and a mesenchymal stem cell are mixed, and the mixture is inoculated to a hydroxyapatite biological ceramic material for culture. In the technical scheme, the mesenchymal stem cell and the chondrocyte are co-cultured according to a certain proportion, proliferation of the chondrocyte can be promoted, and the number of the chondrocytes can be increased. A result shows that the chondrocyte made by adopting the three-dimensional chondrocyte culture method is transplanted to a primary-generation chondrocyte of an injured cartilage in a rear injury model of a rabbit joint injury model, the content of type-II collagen and glycosaminoglycan is obviously increased, and it is indicated that the injured cartilage tissue in the injury model is very well repaired.
Description
Technical field
The invention belongs to cell technology field is and in particular to a kind of three-dimensional culture method of cartilage cell, prepared cartilage
Tissue and application.
Background technology
Articular cartilage damage is the main pathological change of osteoarthropathy, and caused by the reason such as wound, cartilage damage finally also can be led
Cause osteoarthropathy.And do not have after cartilage damage or seldom have self-repairing capability.Human intervention repair of cartilage method is mainly soft
Under bone, bone drilling reparation cartilage defect, cartilage transplantation, periosteum and perichondrium transplanting, chondrocyte cell transplantation and undifferentiated mesenchyma are thin
Born of the same parents' transplanting etc..
The method of the treatment articular cartilage defect of most common of which is cartilaginous tissue transplanting.The method gathers patient's itself
Cartilaginous tissue, isolates cartilage cell, and cartilage cell is bred, and then cartilaginous tissue is transplanted at patient's defect.
Although the method can alleviate cartilage defect to a certain extent, in the method, limited by chondrocyte proliferation, therefore transplanting
Less to internal cartilaginous tissue area, curative effect is not very obvious.
Content of the invention
In view of this, present invention aims to the defect of prior art, provide a kind of three-dimensional training of cartilage cell
Foster method, prepared cartilaginous tissue and application, to promote the propagation of cartilage cell and the reparation of cartilage damage.
For realizing the purpose of the present invention, the present invention adopts the following technical scheme that.
A kind of three-dimensional culture method of cartilage cell, takes cartilage cell and the mixing of adipose stromal stem cell, is inoculated into hydroxyl
Cultivated on apatite bioceramic material.
Preferably, the number of cells of described cartilage cell and described adipose stromal stem cell is than for 4:1.
Preferably, the inoculum concentration of cartilage cell described in described three-dimensional culture method is 2 × 107Individual/hole, fat mesenchymal
The inoculum concentration of stem cell is 0.5 × 107Individual/hole.
Preferably, complex calcium phosphate bioceramic material described in described three-dimensional culture method is the nanoscale of 3D printing
Hydroxyl apatite bioceramic material.
Preferably, described dimensional culture vessel are six orifice plates, each six orifice plates inoculation 3mm × 3mm after cropped ×
0.3mm (length × width × height) nano-grade hydroxy apatite bioceramic material.
Preferably, the condition of culture described in described three-dimensional culture method is 37 DEG C, cultivates under 5% gas concentration lwevel
The DMEM high sugar nutrient solution containing 10% Australia hyclone is added to continue culture after 4h.
Preferably, the time of culture described in described three-dimensional culture method is 40-50 days.
Cartilage cell described in the three-dimensional culture method of cartilage cell of the present invention is preferably primary Autologous Chondrocyte.
In some embodiments, the preparation method of described primary Autologous Chondrocyte specifically includes following steps：1) take
About 3～4cm3Cartilaginous tissue be placed on aseptic plank, shredded to 1mm with aseptic operation knife3Size；
2) above-mentioned cartilaginous tissue fragment is transferred in 50mL centrifuge tube, with the no Ca containing 10% hyclone2+、Mg2+Phosphorus
Hydrochlorate cushioning liquid (PBS solution) washs twice；
3) add II Collagenase Type solution of 2～5 times of volumes of cartilaginous tissue fragment, digestion 4～5 is little on 37 DEG C of shaking tables
When, rotating speed is 100～150rpm；
4) the DMEM culture medium adding the hyclone containing 10% terminates digestion；
5) filtered using 200 eye mesh screens；
6) filtrate centrifugation, after centrifugation, the precipitation of test tube bottom is cartilage cell；
7) cartilage cell is resuspended with the DMEM culture medium of the hyclone containing 10%, according to 1 × 105～2 × 105/ mL's
Density is seeded in T25 blake bottle, cultivates 36h in 37 DEG C, the incubator of 5% carbon dioxide.
Preferably, described adipose stromal stem cell is autologous fat interstital stem cell.
In some embodiments, the preparation method of described third generation umbilical cord mesenchymal stem cells specifically includes following step
Suddenly：1) take adipose tissue 800g centrifugation 10min, suck lower floor's liquid with pasteur pipet, add isopyknic 0.15%I Collagen Type VI
Enzyme, fully mixes.
2) in 37 DEG C of constant temperature gas bath shaking tables, 200R digests 30min, and 800g is centrifuged 10min；
3) remove supernatant, add physiological saline re-suspended cell, with 100 mesh disposable strainer filtering cell；
4) filtrate 800g is centrifuged 10min；
5) remove supernatant, with the DMEM culture medium re-suspended cell containing 10% hyclone, be seeded in the Tissue Culture Flask of T25
In, cultivated in 37 DEG C, the incubator of 5% carbon dioxide.
6), after 24 hours, carry out cell and change liquid；
7), after 36 hours, passage is become P1 to be passed on when cell reaches 80% fusion for cell again, Zhi Daoxi
Born of the same parents' number is 1 × 107～2 × 107Individual.
Present invention also offers the cartilaginous tissue that described preparation method is obtained includes cartilage cell and adipose stromal is done carefully
Born of the same parents and hydroxyl apatite bioceramic material.Cartilage cell and adipose stromal stem cell are trained in hydroxyl apatite bioceramic altogether
Support.
The present invention adopts the impact to rabbit joint injury for the above-mentioned cartilaginous tissue of rabbit joint injury model inspection, and result shows warp
II Collagen Type VI in the Primary chondrocyte of damaged cartilage in damage model after cartilaginous tissue reparation of the present invention and osamine
Polyoses content is significantly raised, shows that in damage model, damaged cartilage tissue has obtained fine reparation.
Therefore present invention also offers cartilaginous tissue treats the application in the product that cartilage damage is repaired in preparation.
Compared with prior art, the invention has the advantages that one of：
(1) co-cultured by a certain percentage with cartilage cell using adipose stromal stem cell, can be promoted cartilage cell's
Propagation, improves the quantity of cartilage cell；
(2) cartilage cell in the present invention and adipose stromal stem cell are all from autologous, no immunological rejection effect after transplanting,
Security is of a relatively high；
(3) the various biomechanical properties of the nano-grade hydroxy apatite bioceramic of 3D printing more conform to hard group of human body
The requirement knitted, is more beneficial for cartilage damage reparation.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing having required use in technology description is briefly described.
Fig. 1 shows the form (100 times) of Primary chondrocyte.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work, all
Belong to the scope of protection of the invention.
For a further understanding of the present invention, with reference to specific embodiment, the present invention will be described in detail.As no special
Illustrate, experiment material involved in the present invention and reagent all can be bought by commercial channel and obtain.The nanometer of wherein 3D printing
Level hydroxyl apatite bioceramic is provided by South China Science ＆ Engineering University.
Embodiment 1
1st, the primary separation of rabbit Autologous Chondrocyte
1) rabbit knee cartilage is taken to organize 3～4cm3, in super-clean bench, cartilaginous tissue is placed on aseptic plank, with aseptic
Knife blade shreds cartilaginous tissue to 1mm3The size of left and right；
2) cartilaginous tissue after chopping is transferred in 50ml centrifuge tube (CORNING), with the nothing containing 10% hyclone
Ca2+、Mg2+PBS (PBS solution) washs twice；
3) add II Collagenase Type solution of 2～5 times of volumes of cartilaginous tissue, with 100～150rpm's on 37 DEG C of shaking tables
Rotating speed digests 4～5 hours.
4) the DMEM culture medium adding the hyclone containing 10% terminates digestion；
5) and then by the digestive juice screen filtration of 200 mesh；
6) filtrate is centrifuged, after centrifugation, the precipitation of test tube bottom is cartilage cell；
7) cartilage cell is resuspended with the DMEM culture medium of the hyclone containing 10%, according to 1 × 105/ ml～2 × 105/
The density of ml is seeded in T25 blake bottle (CORNING), is cultivated in 37 DEG C, the incubator of 5% carbon dioxide.36 is little
Shi Hou, observes primary Autologous Chondrocyte under an optical microscope.Result such as Fig. 1.Carry out again when cell reaches 80% fusion
Pass on, until cell number is 1 × 107～2 × 107Individual.
The form that Fig. 1 is shown in the primary Autologous Chondrocyte under light microscope (100 times) is in paving stone shape.
2nd, the primary separation of rabbit autologous fat mescenchymal stem cell and Secondary Culture
1) extract rabbit abdominal adipose tissue 10ml；
2) 800g centrifugation 10min, sucks lower floor's liquid with pasteur pipet, adds isopyknic 0.15%I Collagenase Type, fills
Divide and mix.
3) in 37 DEG C of constant temperature gas bath shaking tables, 200R digests 30min, and 800g is centrifuged 10min；
4) remove supernatant, add physiological saline re-suspended cell, with 100 mesh disposable strainer filtering cell；
5) filtrate 800g is centrifuged 10min；
6) remove supernatant, with the DMEM culture medium re-suspended cell containing 10% hyclone, be seeded in the Tissue Culture Flask of T25
In, cultivated in 37 DEG C, the incubator of 5% carbon dioxide.
7), after 24 hours, carry out cell and change liquid；
8), after 36 hours, passage is become P1 for cell.
9) passed on again when cell reaches 80% fusion, until cell number is 1 × 107～2 × 107Individual.
3rd, dimensional culture
1) after ADSCs normal saline flushing being passed on, the EDTA digestion of the trypsase+0.01% with 0.05%, from
Remove supernatant after the heart, be prepared into 1 × 106The cell suspension of/ml.
2) by after passaged chondrocytes normal saline flushing, the EDTA of the trypsase+0.01% with 0.05% digests,
Remove supernatant after centrifugation, be prepared into 1 × 106The cell suspension of/ml.
3) with every hole 1ml respectively by above two cell suspension according to 1:4 volume ratio ratio (ADSCs：Cartilage cell)
Drop in the surface of the nano-grade hydroxy apatite bioceramic material of 3D printing, enter the dioxy that 37 DEG C of gas concentration lwevels are 5%
After change carbon incubator is cultivated 4 hours, then plus the high sugar nutrient solution 1ml continuation culture 40-50 of the DMEM containing 10% Australia hyclone
My god.
Embodiment 2 neocartilage tissue repair rabbit joint injury
1) set up rabbit joint injury model；
2) cartilaginous tissue cultivating embodiment 1 is transplanted to injury region, repaiies respectively at Post operation 12 weeks, 24 weeks collecting parts
Cartilaginous tissue (reparation group) after multiple and the damaged cartilage not transplanting cartilaginous tissue (control group), separate Primary chondrocyte, inspection
Survey the expression of II Collagen Type VI in the case of two groups and glycosaminoglycan, result such as table 1.
Table 1 II Collagen Type VI and the expression of glycosaminoglycan
As shown in Table 1, the II Collagen Type VI content that 12 weeks after operation tests reparation group tissue is 11.50 μ g/ml, and control group is
8.92 μ g/ml, II Collagen Type VI content of postoperative 24 weeks experiment reparation group tissues is 19.87 μ g/ml, and control group is 13.64 μ g/
ml；The GAG content that 12 weeks after operation tests reparation group tissue is 6.54 μ g/ml, and control group is 2.46 μ g/ml, experiment in postoperative 24 weeks
The GAG content of reparation group tissue is 18.30 μ g/ml, and control group is 4.45 μ g/ml.Illustrate that damaged cartilage is repaired very well.
Claims (11)
1. a kind of three-dimensional culture method of cartilage cell, it is characterised in that taking cartilage cell and the mixing of adipose stromal stem cell, connects
Plant and cultivated on hydroxyl apatite bioceramic material.
2. three-dimensional culture method according to claim 1 is it is characterised in that described cartilage cell and described adipose stromal are done
The number of cells of cell is than for 4:1.
3. three-dimensional culture method according to claim 1 is it is characterised in that the inoculum concentration of described cartilage cell is 2 × 107
Individual/hole, the inoculum concentration of fat mesenchymal stem cell is 0.5 × 107Individual/hole.
4. three-dimensional culture method according to claim 1 is it is characterised in that described complex calcium phosphate bioceramic material is
The nano-grade hydroxy apatite bioceramic material of 3D printing.
5. three-dimensional culture method according to claim 1 is it is characterised in that described dimensional culture vessel are six orifice plates,
Each six orifice plate inoculations 3mm × 3mm × 0.3mm nano-grade hydroxy apatite bioceramic material after cropped.
6. three-dimensional culture method according to claim 1 is it is characterised in that described culture is 37 DEG C of 5% dense carbon dioxide
The DMEM high sugar nutrient solution continuation culture containing 10% Australia hyclone is added after degree lower culture 4h.
7. three-dimensional culture method according to claim 1 is it is characterised in that the time of described culture is 40-50 days.
8. three-dimensional culture method according to claim 1 is it is characterised in that described cartilage cell is that primary autologous cartilage is thin
Born of the same parents.
9. three-dimensional culture method according to claim 1 is it is characterised in that described adipose stromal stem cell is autologous fat
Interstital stem cell.
10. the cartilaginous tissue that preparation method described in claim 1-8 any one is obtained.
Application in the product that preparation treatment cartilage damage is repaired for the cartilaginous tissue described in 11. claims 9.
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