CN101748095B - Method for directionally inducing cartilage cells - Google Patents
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- CN101748095B CN101748095B CN 200810203814 CN200810203814A CN101748095B CN 101748095 B CN101748095 B CN 101748095B CN 200810203814 CN200810203814 CN 200810203814 CN 200810203814 A CN200810203814 A CN 200810203814A CN 101748095 B CN101748095 B CN 101748095B
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Abstract
The invention belongs to the technical field of biology and relates to a method for directionally inducing cartilage cells, in particular to the method for directionally inducing cartilage cells by human amniotic epithelial cells. The method comprises the following steps: performing primary culture and subculture on the amniotic epithelial cells through in vitro culture of the human amniotic epithelial cells according to a tissue engineering genomics principle; observing and adjusting a genotype of the amniotic epithelial cells; inducing. regulating and controlling the amniotic epithelial cells by a cell factor (a transforming growth factor beta, TGF-beta) and BMP-7 to split the amniotic epithelial cells into the cartilage cells through directional induction; detecting the cartilage cell property and the cartilage cell activity of the induced and split cells by a molecular biology method; determining a regulating and controlling mechanism for directionally inducing the cartilage cells by the human amniotic epithelial cells; and obtaining a result which indicates that the human amniotic epithelial cells can be used as a seed source for inducing and splitting the cartilage cells. The method gets materials conveniently and has relatively wide application prospect.
Description
Technical field
The invention belongs to biological technical field.A kind of method that relates to directionally inducing cartilage cells is specifically related to a kind of method that adopts human amniotic membrane epithelial cell directionally inducing cartilage cells.
Background technology
The research report, the cartilage injury that arthrotrauma and degenerative change etc. causes can cause the long-term pain in joint and dysfunction, and even the useless use in joint, bring great misery and inconvenience to patient's life.Joint cartilage is without blood supply and innervation, almost can not spontaneously repair after damage, the spontaneous reaction of repairing can occur in some cases, but newborn tissue is the fibrous cartilage tissue, be very different aspect weave construction and biological chemistry with normal hyaline cartilage, and under physiological stress, degenerative change can occur, finally develop into osteoarthritis.Traditional methods for the treatment of, as boring under little fracture, cartilage, cartilage and perichondrium transplanting etc., the symptoms such as alleviating pain that can only be temporary transient all can not be successfully completed the reparation of hyaline cartilage, and under cartilage wherein, boring also is only applicable to the little case of cartilage injury face; Transplant from body cartilaginous tissue, perichondrium, need second operation, and can cause the position damage of drawing materials; Artificial total joint prosthetic replacement cost is high, complication is many, and is not suitable for young patient.Along with the development of life science and engineering science, there is research and utilization engineering science ways and means to build and regenerate various tissues and the alternative disease damage tissue of organ, and obtained significant achievement, for new approach is opened up in the treatment of cartilage disease.Build the biological nature tissue engineering bone/cartilage similar to the natural joint cartilage and become one of focus of present Tissue Engineering Study.
Prior art discloses about cartilage tissue engineered 3 key elements: a quantity of seeds cell, suitable timbering material and the optimization of condition of in vitro culture.Articular cartilage depends on the selection of seed cell to a great extent.The seed cell that at present research is used for tissue engineering bone/cartilage has chondrocyte, embryo source sexual cell, mescenchymal stem cell, periosteum cell, synovial cell etc.
Confirm that according to foreign study embryo source sexual cell (embryonic stem cell) has stronger multiplication capacity than the chondrocyte of adult system, and do not cause obvious allosome rejection, with it as the good effect of the damaged acquisition of seed cell repairing articular cartilage, repair tissue also has no obvious regression, but its source and the ethics problem followed thereof are to limit the greatest problem of its application.People's amniotic epithelial cells (human amniotic epithelial cells, HAECs) derive from and normally cut open discarded fetal membrane in palace postpartum, do not destroy fetal tissues, need not in the fetal development of embryo's premature termination, do not exist and cause the Medical Ethics dispute, people's amniotic epithelial cells has the multi-lineage potential of pre-embryo stem cell simultaneously, amniotic epithelial cells is also seldom expressed the antigens such as HLA-A, B, C or DR, be difficult for after transplanting producing immunological rejection, promise to be the cartilage tissue engineered seed cell source that builds.The prior art relevant to present method and cartilage tissue engineered experimental principle have following reference:
1?Kawaguchi?J，Mee?PJ，Smith?AG.Osteogenic?and?chondrogenic?differentiation?of?embryonic?stemcells?in?response?to?specific?growth?factors.Bone.2005，36(5)：758-769
2?Gan?L，Kandel?RA.In?vitro?cartilage?tissue?formation?by?Co-culture?of?primary?and?passagedchondrocytes.Tissue?Eng.2007，13(4)：831-842
3?Afizah?H，Yang?Z，Hui?JH.A?comparison?between?the?chondrogenic?potential?of?human?bone?marrowstem?cells(BMSCs)and?adipose-derived?stem?cells(ADSCs)taken?from?the?same?donors.Tissue?Eng.2007，13(4)：659-666
4?Sudo?K，Kanno?M，Miharada?K.Mesenchymal?progenitors?able?to?differentiate?into?osteogenic，chondrogenic，and/or?adipogenic?cells?in?vitro?are?present?in?most?primary?fibroblast-like?cellpopulations.Stem?Cells.2007，25(7)：1610-1617
5?Nakamura?Y，Wang?X，Xu?C.Xenotransplantation?of?long-term-cultured?swine?bone?marrow-derivedmesenchymal?stem?cells.Stem?Cells.2007，25(3)：612-620
6?Karahuseyinoglu?S，Cinar?O，Kilic?E.Biology?of?stem?cells?in?human?umbilical?cord?stroma：in?situand?in?vitro?surveys.Stem?Cells.2007，25(2)：319-331
7?Christley?S，Alber?MS，Newman?SA.Patterns?of?mesenchymal?condensation?in?a?multiscale，discretestochastic?model.PLoS?Comput?Biol.2007，3(4)：e76
8?Derfoul?A，Miyoshi?AD，Freeman?DE.Glucosamine?promotes?chondrogenic?phenotype?in?bothchondrocytes?and?mesenchymal?stem?cells?and?inhibits?MMP-13?expression?and?matrix?degradation.Osteoarthritis?Cartilage.2007，15(6)：646-655
9?Song?YS，Ku?JH.Monitoring?transplanted?human?mesenchymal?stem?cells?in?rat?and?rabbit?bladdersusing?molecular?magnetic?resonance?imaging.Neurourol?Urodyn.2007，26(4)：584-593
10?Narukawa?M，Suzuki?N，Takayama?T.Enamel?matrix?derivative?stimulates?chondrogenicdifferentiation?of?ATDC5?cells.J?Periodontal?Res.2007，42(2)：131-137
11?Chao?PH，Grayson?W，Vunjak?Novakovic?G.Engineering?cartilage?and?bone?using?humanmesenchymal?stem?cells.J?Orthop?Sci.2007，12(4)：398-404
12?Hachisuka?H，Mochizuki?Y，Yasunaga?Y.Flow?cytometric?discrimination?of?mesenchymal?progenitorcells?from?bone?marrow-adherent?cell?populations?using?CD34/44/45(-)and?Sca-1(+)markers.JOrthop?Sci.2007，12(2)：161-169
13?Shintani?N，Kurth?T，Hunziker?EB.Expression?of?cartilage-related?genes?in?bovine?synovial?tissue.JOrthop?Res.2007，25(6)：813-819
14?Karlsson?C，Brantsing?C，Svensson?T.Differentiation?of?human?mesenchymal?stem?cells?and?articularchondrocytes：analysis?of?chondrogenic?potential?and?expression?pattern?of?differentiation-related?transcription?factors.J?Orthop?Res.2007，25(2)：152-163
15?Eyrich?D，Wiese?H，Maier?G..In?vitro?and?in?vivo?cartilage?engineering?using?a?combination?ofchondrocyte-seeded?long-term?stable?fibrin?gels?and?polycaprolactone-based?polyurethane?scaffolds.Tissue?Eng.2007，13(9)：2207-2218
16?Hwang?NS，Varghese?S，Puleo?C.Morphogenetic?signals?from?chondrocytes?promote?chondrogenicand?osteogenic?differentiation?of?mesenchymal?stem?cells.J?Cell?Physiol.2007，212(2)：281-284
17?Song?JJ，Aswad?R，Kanaan?RA.Connective?tissue?growth?factor(CTGF)acts?as?a?downstreammediator?of?TGF-betal?to?induce?mesenchymal?cell?condensation.J?Cell?Physiol.2007，210(2)：398-410
Summary of the invention:
A kind of method that the purpose of this invention is to provide directionally inducing cartilage cells is specifically related to a kind of method that adopts human amniotic membrane epithelial cell directionally inducing cartilage cells.
The present invention utilizes the organizational engineering method, adopt human amniotic membrane epithelial cell (human amniotic epithelialcells, HAECs) as cartilage tissue engineered seed cell, build the tissue engineering bone/cartilage similar to the natural joint cartilage with the regeneration biological characteristic.
the present invention is according to organizational project genetics principle, by the vitro culture of human amniotic epithelial cells, carry out the primary of amniotic epithelial cells, the cultivation of going down to posterity, observe and adjust the amniotic epithelial cells genotype, utilize cytokine (transforming growth factor-beta, TGF-β) and BMP-7 induce and regulate and control its Induction of committed differentiation chondroblast, the chondrocyte's characteristic and the chondrocyte that detect the cell of inducing differentiation by molecular biology method are active, the regulatory mechanism of clear and definite people's amniotic epithelial cells directionally inducing cartilage cells, result shows that people's amniotic epithelial cells can be used as the seed source of chondrogenic differentiation.Present method provides theory and practice basic for cartilage tissue engineered application.
The inventive method comprises the steps:
1) external primary, the cultivation of going down to posterity of people's amniotic epithelial cells;
2) regulation and control people's amniotic epithelial cells (embryonic stem cell-like) genotype utilizes cytokine (transforming growth factor-beta, TGF-β) and BMP-7 to intervene the regulation and control directionally inducing cartilage cells;
3) chondrocyte's determination of activity,
Chondrocyte's characteristic and the anti-apoptosis characteristic of the cell of differentiation induced in detection;
4) induce the transcriptional activity that passes through to depend on transcription factor Sox9 of differentiation regulation and control to act on II Collagen Type VI Col2a enhancer sequence, in the common presence of CBP/p300, make the up-regulated of Col2a1, the initial chondrocyte's of test differentiation signal conduction path.
Chondrocyte's electron microscopic observation result after inducing shows, induces the rounded or Polygons of rear chondrocyte, and cytolemma is complete, is fan clam sample projection, the tenuigenin uniformity, and as seen differ in size cavity and lysosome, the cytoplasm Mitochondria is less, and nucleus is complete.
Seed Cells of Tissue Engineering Cartilage of the present invention-people's amniotic epithelial cells derives from normal postcesarean fetal membrane.
The described amniotic epithelial cells of present method originates from morular ectoderm cell group in fetal development, it is the early stage product of fetal development due to amnion, amniotic epithelial cells has the multi-lineage potential of pre-embryo stem cell, seldom express simultaneously human tissue human leucocyte antigen HLA-A, B, C or DR antigen, be difficult for producing immunological rejection after transplanting, the present invention has adopted specific human amniotic membrane epithelial cell as Seed Cells of Tissue Engineering Cartilage, draw materials conveniently, have more wide prospect, be expected to become the cartilage tissue engineered seed cell source that builds.
Description of drawings
Fig. 1 be people's amniotic epithelial cells external primary/go down to posterity cultivate that phase microscope observes that HAECs sticks, growth, propagation situation,
Wherein, A: cultivate the human amniotic membrane epithelial cell of 2nd generation, B: the human amniotic membrane epithelial cell of cultivating for the 4th generation.
Fig. 2 observes under inverted microscope to induce chondrocyte's form.
Fig. 3 is that chondrocyte's form is induced in 1% Toluidine blue staining observation.
Fig. 4 is that the observation of immunohistochemical methods fluorescent test induces the chondrocyte active.
Fig. 5 induces rear chondrocyte's electron microscopic observation result.
Embodiment
Embodiment 1
(1) external primary, the cultivation of going down to posterity of people's amniotic epithelial cells
Fetal membrane sample 12 examples of employing after cesarean section is discarded according to a conventional method with amnion and tapetum blunt separation, are placed in the PBS that contains two anti-(100 μ/ml penicillin, 100 μ g/ml Streptomycin sulphates) and repeatedly rinse.Amnion after rinsing is cut into approximately 1mm * 1mm fritter with eye scissors, add 0.2% collagenase at the 37 ℃ of standing digestion of incubator 2h, the centrifugal 5min of 1000r/min, sucking-off collagenase digesting liquid, then add 0.25% tryptic digestive juice at room temperature to digest 15min, stop digestion with the substratum that contains serum, use again 200 purpose screen filtrations, the collecting cell suspension, the centrifugal 5min of 1000r/min is with 1 * 10
5The amniotic epithelial cells 2ml cell of individual/ml density is inoculated in culturing bottle, puts in 5%CO2,37 ℃ of incubators and cultivates, and changes every other day liquid 1 time, and general 5～7d went down to posterity 1 time with 1: 3.Growth and proliferation of cell to 80% will in time go down to posterity when merging.Exhaust substratum, add 0.25% tryptic digestive juice in culturing bottle, 37 ℃ of digestion 5min.See the kytoplasm retraction under phase microscope, stop digestion after the intercellular substance increases, absorb Digestive system, add the substratum that contains serum to end digestion, piping and druming, make cell suspension gently, after cell counting, in 1: 2～3 ratio inoculation culture, culture condition is the same, changes every other day liquid.
The cell growth curve result shows:
Primary postvaccinal 2～6d is the latent period of people's amniotic epithelial cells (HAECs) growth, and this phase is mainly the adherent growth stage of HAECs, and the propagation of culturing cell is very not active; Beginning in the 7th day, under inverted microscope, visible attached cell forms a plurality of cell clones not of uniform size, shows that the propagation of cell this moment begins to enliven; Those cell clones further enlarged in the 7th～9 day, and many cell clones are connected with each other, and cell growth curve shows that this phase cell number is exponential and increases progressively, and this phase is the logarithmic proliferation phase of the primary incubation growth of HAECs; The 10th～14 day, the connected also major part of bottom surface, confluent culture hole of cell clone, cell growth curve shows that HAECs grows into a plateau; Go down to posterity to cultivate and be about 24～36h latent period; Go down to posterity and cultivate the logarithmic proliferation phase and be about 4～6d; After the logarithmic proliferation phase finishes, to the rear 8-9d of inoculation, the HAECs growth is slow gradually, enters plateau.
As shown in Figure 1, the human amniotic membrane epithelial cell through vitro culture primary/go down to posterity cultivate after 200 times of light microscopics take the photograph sheet, see as shown go down to posterity the 4th generation cell breeding cycle breeding potential better, have adherent growth ability preferably, the attached cell volume is larger, is consistent spindle cell.Through three all serum-free medium inducing culture, the part cell begins to become broad, smooth Polygons and many pseudopodium stretch the shape cell, and the parts of fine dysuria with lower abdominal colic that continues becomes circle or ellipse, and cell peripheral has been secreted a large amount of matrix.
(2) regulation and control people's amniotic epithelial cells (embryonic stem cell-like) genotype, directionally inducing cartilage cells
Adjust people's amniotic epithelial cells (embryonic stem cell-like) genotype, induce and control it and break up to the chondroblast direction, the specific albumen of described people's amniotic epithelial cells transfection or somatomedin, particularly affect into the transcription factor of cartilage differentiation, as Sox family (SRY-type high mobility group box family): Sox5, Sox6, Sox8, Sox9; Lc-Maf (long form of the c-Maf transcription factor), CREB is in conjunction with albumen [CREB-binding protein (CBP/p300)], Nkx3.2, some members of Pax9 (paired-box containing protein) and Smad family can cause cartilage and occur.Ways of regeneration by the mediation of the signals such as TGF-β, its molecular mechanism relates to Sox-9 transcriptional control signal transduction pathway, and act on II Collagen Type VI Col2a1 enhancer sequence by the transcriptional activity that depends on transcription factor Sox9, in the common presence of CBP/p300, make the up-regulated of Col2a1, initial chondrocyte's differentiation.
Present method is preferably utilized transforming growth factor TGF-β and bone forming generation protein-7 (bone morphogenetic proteins-7, BMP-7) as inducible factor, the II Collagen Type VI is fixed into fibroblast growth factor (fibroblast growth factors, FGF) and rhIGF-1 (Insulin-like growth factor, IGF) form cultivating timbering material carries out human amniotic membrane epithelial cell (human amniotic epithelial cells, HAECs) vitro culture and induces the chondrocyte.
people's amniotic epithelial cells (HAECs) suspension that goes down to posterity is inoculated in 6 well culture plates of inserting in advance cover glass with 1/ml, diameter 3.5cm, each hole adds the 2mlH-DMEM complete culture solution, be changed to 2mlH-DMEM serum-free inducing culture liquid next day, wherein contain respectively transforming growth factor β_1 0ng/ml, bone forming generation protein-7 (bone morphogenetic proteins-7, BMP-7) 1 μ g/ml, dexamethasone 100nMol/L, vitamins C 50 μ g/ml, Regular Insulin 10U/ml, thyroxine 50ng/ml, sodium β-glycerophosphate 20mMol/L and bovine serum albumin 1.25mg/ml, only add the complete H-DMEM nutrient solution of 2ml to cultivate in control wells, the next day change liquid once.After inducing the 7th, 14,21 day respectively, take out cover glass, carry out observation of cell form (seeing Fig. 2) under corresponding detection-inverted microscope, 1% Toluidine blue staining (seeing Fig. 3), immunohistochemical staining (seeing Fig. 4), electron microscopic observation cell appearance and matrix (seeing Fig. 5).
Result shows, described TGF-β 1 is external respectively 1,10,50, can induce HAECs to the differentiation of cartilage direction during 100mg/L, but effect is best when 10mg/L; The effect of described somatomedin has dose-dependently within the specific limits, exceeds this described scope effect not obvious; The chondrocyte has the acceptor of multiple somatomedin, different somatomedins acts on different growth cycles, a kind of somatomedin can be regulated expression and the activity of other somatomedins, can not induce HAECs to break up to the cartilage direction as BMP-7 or the independent application of IGF-1, but when share with TGF-β 3, more alone TGF-β 3 can break up to the cartilage direction by the more effective HAECs that induces.
(3) the chondrocyte's characteristic, chondrocyte proliferation activity, the anti-apoptosis capacity that detect the cell of inducing differentiation adopt respectively following detection method and index:
Adopt that phase microscope observes that HAECs sticks, growth, propagation situation;
Adopt and observe chondrocyte's Histological Study, propagation situation under scanning electronic microscope;
Adopt the active chondrocyte's survival of MTT colorimetric determination and multiplication capacity;
Adopt 3H-Proline to measure the chondrocyte active, qualitatively judge and detection by quantitative;
Adopt the TUNEL staining to carry out the detection of articular chondrocyte apoptosis in cartilaginous tissue;
Adopt RT-PCR to detect the expression of Caspase-3mRNA sequence;
Adopt elisa assay Caspase-3 protease activity;
Adopt RT-PCR to detect the expression of MMP-1 and MMP-13;
Detect the Bcl-2 protein expression.
Described RT-PCR reacts by following step:
(1) extraction of total RNA: get the inboard cartilaginous tissue of the interior condyle of femur and put homogenate in 1ml Trizol liquid, the step of by specification is extracted total RNA;
(2) reverse transcription: reaction volume is 50 μ l: total RNA 1 μ g, turn ice chest after 70 ℃ of sex change 5min, add 50mg/L random primer 1 μ l, RNasin 1 μ l, 10mmol/L dNTP 2 μ l, 5 * buffer, 10 μ l, RTase1 μ l, mend to 50 μ l without RNA enzyme water, after 42 ℃ of 60min, 95 ℃ of 5min termination reactions;
(3) pcr amplification: reaction volume 50 μ l, 10mmol/L dNTP 1 μ l, 25mmol/L MgCl2 4 μ l, 10 * buffer, 5 μ l, 3 ' premier 0.5 μ l, 5 ' premier 0.5 μ l, cDNA 4 μ l, Taq enzyme 2U, deionized water complements to 50 μ l, 95 ℃ of denaturation 5min, 95 ℃ of sex change 45s, at MMP-1:55 ℃, MMP-3:57 ℃, TIMP-1:60 ℃, the annealing temperature 45s of GAPDH:57 ℃, cyclic amplification under the condition of 72 ℃ of extension 45s, wherein capable 30 circulations of GAPDH, MMP-1,-3 and capable 35 circulations of TIMP-1, amplified production is electrophoresis on 1.5% sepharose, observe the amplified production specific band under ultraviolet transilluminator,
(4) product carries out image analysis through French VL gel imaging and analytical system, measure absorbancy (A) value of band, make internal reference with GAPDH, use MMP-1 ,-3 and the A value of TIMP-1 band respectively with the relative level of its expression of ratio value representation of the A value of GAPDH.
(4) induce the research of differentiation regulation and control Sox-9 transcriptional control signal transduction pathway
The immunoenzyme joint-trial is tested, Westernbolt Protein Detection Sox-9 protein expression, the variation of clear and definite its expression level cell after inducing differentiation, clear and definite TGF-β, Sox-9 regulating and controlling effect are in II Collagen Type VI Col2a1 enhancer sequence, in the common presence of CBP/p300, make the up-regulated of Col2a1, the mechanism of action of initial chondrocyte's differentiation signal conduction path.
Claims (3)
1. the method for a directionally inducing cartilage cells, is characterized in that adopting the human amniotic membrane epithelial cell as seed cell, by following step, and directionally inducing cartilage cells:
1) external primary, the cultivation of going down to posterity of people's amniotic epithelial cells, wherein,
Amnion after separation is placed in the PBS that contains 100 μ/ml penicillin and 100 μ g/ml Streptomycin sulphates and rinses, and adds 0.2% collagenase, 37 ℃ of incubator digestion 2h, 1000r/min is centrifugal, the sucking-off Digestive system, add 0.25% tryptic digestion 15min, stop digestion with the substratum that contains serum, 200 eye mesh screens filter, and 1000r/min is centrifugal for the collecting cell suspension, cell is inoculated in culturing bottle, put in 5%CO2,37 ℃ of incubators and cultivate, change every other day liquid, went down to posterity 1 time with 1: 3 in 5～7 days; Growth and proliferation of cell to 80% merges, and exhausts substratum, adds 0.25% tryptic digestive juice, 37 ℃ of digestion 5min; Absorb Digestive system, add the substratum that contains serum to end digestion, cell suspension processed changes liquid every other day in 1: 2～3 ratio inoculation culture;
2) regulation and control people amniotic epithelial cells genotype, directionally inducing cartilage cells, wherein,
Utilize transforming growth factor-beta and bone forming generation protein-7 as inducible factor, the II Collagen Type VI is fixed into fibroblast growth factor and rhIGF-1 and forms and cultivate timbering material and carry out human amniotic membrane epithelial cell vitro culture and induce the chondrocyte, comprises step:
The people's amniotic epithelial cells suspension that goes down to posterity is inoculated in 6 well culture plates of inserting in advance cover glass with 1ml, each hole adds the 2mlH-DMEM complete culture solution, is changed to 2mlH-DMEM serum-free inducing culture liquid next day,
Contain transforming growth factor β_1 0ng/ml, bone forming generation protein-7 1 μ g/ml, dexamethasone 100nMol/L, vitamins C 50 μ g/ml, Regular Insulin 10U/ml, thyroxine 50ng/ml, sodium β-glycerophosphate 20mMol/L and bovine serum albumin 1.25mg/ml in described serum-free inducing culture liquid;
3) chondrocyte's determination of activity, chondrocyte's characteristic and the anti-apoptosis characteristic of the cell of differentiation induced in detection.
2. by the method for directionally inducing cartilage cells claimed in claim 1, it is characterized in that described people's amniotic epithelial cells is embryonic stem cell-like.
3. by the method for directionally inducing cartilage cells claimed in claim 3, it is characterized in that, the described cell that is inoculated in culturing bottle is 1 * 10
5The amniotic epithelial cells of individual/ml density.
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| CN102925408A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Method for differentiating induced pluripotent stem (iPS) cells into cartilage cells |
| CN104371971A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Method for obtaining amniotic epithelial cells through separation |
| CN103638558B (en) * | 2013-09-30 | 2015-04-29 | 中国人民解放军第三军医大学第二附属医院 | In vitro construction method for bionic ligament-bone tissue engineering connector |
| CN103966159B (en) * | 2014-02-13 | 2015-08-05 | 天津和泽干细胞科技有限公司 | Human plactnta Subaerial blue green algae and stem cell bank construction process thereof |
| CN106434539A (en) * | 2016-09-30 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Osteogenic induction medium and osteogenic differentiation method |
| CN108060122B (en) * | 2016-11-07 | 2021-07-09 | 深圳臻德济慈药品研发有限公司 | Small molecule compound combination and method for preparing chondrocytes by using cells induced to differentiate by small molecule compound combination |
| AU2020252087A1 (en) * | 2019-03-29 | 2021-11-18 | Kolon Tissuegene, Inc. | Mixed-cell gene therapy |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661010A (en) * | 2004-02-25 | 2005-08-31 | 上海组织工程研究与开发中心 | Method for inducing fibroblast to form cartilage cells |
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Non-Patent Citations (2)
| Title |
|---|
| GROWTH FACTOR COMBINATION FOR CHONDROGENIC INDUCTION FROM HUMAN MESENCHYMAL STEM CELL;NITAYA INDRAWATTNAN, ET AL;《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》;20041231;第320卷;914-919 * |
| 人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能;宋秀军，等;《中国组织工程研究与临床康复》;20071216;第11卷(第50期);10056-10058 * |
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