CN100482784C - Method for inducing fibroblast to form cartilage cells - Google Patents
Method for inducing fibroblast to form cartilage cells Download PDFInfo
- Publication number
- CN100482784C CN100482784C CNB2004100165142A CN200410016514A CN100482784C CN 100482784 C CN100482784 C CN 100482784C CN B2004100165142 A CNB2004100165142 A CN B2004100165142A CN 200410016514 A CN200410016514 A CN 200410016514A CN 100482784 C CN100482784 C CN 100482784C
- Authority
- CN
- China
- Prior art keywords
- inoblast
- chondrocyte
- cell
- cartilage
- days
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims abstract description 47
- 230000001939 inductive effect Effects 0.000 title abstract description 23
- 210000002950 fibroblast Anatomy 0.000 title abstract description 14
- 210000003321 cartilage cell Anatomy 0.000 title abstract description 7
- 210000000845 cartilage Anatomy 0.000 claims abstract description 56
- 210000004027 cell Anatomy 0.000 claims description 83
- 210000001612 chondrocyte Anatomy 0.000 claims description 73
- 239000000463 material Substances 0.000 claims description 45
- 235000015097 nutrients Nutrition 0.000 claims description 17
- 230000012010 growth Effects 0.000 claims description 15
- 230000003328 fibroblastic effect Effects 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 2
- 244000309466 calf Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract description 11
- 239000007788 liquid Substances 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 9
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 210000001519 tissue Anatomy 0.000 description 31
- 102000002734 Collagen Type VI Human genes 0.000 description 29
- 108010043741 Collagen Type VI Proteins 0.000 description 29
- 102000016284 Aggrecans Human genes 0.000 description 18
- 108010067219 Aggrecans Proteins 0.000 description 18
- 102000008186 Collagen Human genes 0.000 description 17
- 108010035532 Collagen Proteins 0.000 description 17
- 229920001436 collagen Polymers 0.000 description 17
- 239000011159 matrix material Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 230000004069 differentiation Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 230000008520 organization Effects 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 238000003757 reverse transcription PCR Methods 0.000 description 11
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 9
- 102000013275 Somatomedins Human genes 0.000 description 9
- 210000000988 bone and bone Anatomy 0.000 description 9
- 230000001413 cellular effect Effects 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000006698 induction Effects 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 230000007547 defect Effects 0.000 description 7
- 230000002500 effect on skin Effects 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 102100035379 Growth/differentiation factor 5 Human genes 0.000 description 6
- 229920000954 Polyglycolide Polymers 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000001185 bone marrow Anatomy 0.000 description 6
- 229920000747 poly(lactic acid) Polymers 0.000 description 6
- -1 poly(lactic acid) Polymers 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000012620 biological material Substances 0.000 description 5
- 235000010410 calcium alginate Nutrition 0.000 description 5
- 229960002681 calcium alginate Drugs 0.000 description 5
- 239000000648 calcium alginate Substances 0.000 description 5
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 5
- 238000004264 monolayer culture Methods 0.000 description 5
- 239000004626 polylactic acid Substances 0.000 description 5
- 239000002356 single layer Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 description 4
- 108010090254 Growth Differentiation Factor 5 Proteins 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229920001983 poloxamer Polymers 0.000 description 4
- 239000004633 polyglycolic acid Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 4
- 229920002101 Chitin Polymers 0.000 description 3
- 229920001661 Chitosan Polymers 0.000 description 3
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 3
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 3
- 108010050808 Procollagen Proteins 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 210000004207 dermis Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 3
- 230000011164 ossification Effects 0.000 description 3
- 239000005015 poly(hydroxybutyrate) Substances 0.000 description 3
- 229920000515 polycarbonate Polymers 0.000 description 3
- 239000004417 polycarbonate Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000004593 Epoxy Substances 0.000 description 2
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 2
- 101001023988 Homo sapiens Growth/differentiation factor 5 Proteins 0.000 description 2
- 241001062009 Indigofera Species 0.000 description 2
- 102000055008 Matrilin Proteins Human genes 0.000 description 2
- 108010072582 Matrilin Proteins Proteins 0.000 description 2
- 101710167839 Morphogenetic protein Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102000016611 Proteoglycans Human genes 0.000 description 2
- 108010067787 Proteoglycans Proteins 0.000 description 2
- 150000008065 acid anhydrides Chemical class 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000001188 articular cartilage Anatomy 0.000 description 2
- 238000000889 atomisation Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000022159 cartilage development Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000024835 cytogamy Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical group O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 2
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000011343 solid material Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- JYGRAOYMDDFOSM-FQJIPJFPSA-N (4s)-4-[[(2s)-4-carboxy-2-[[(2s)-3-carboxy-2-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]propanoyl]amino]butanoyl]amino]-5-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-5-oxopentano Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN JYGRAOYMDDFOSM-FQJIPJFPSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 108010049974 Bone Morphogenetic Protein 6 Proteins 0.000 description 1
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102100035368 Growth/differentiation factor 6 Human genes 0.000 description 1
- 101001023964 Homo sapiens Growth/differentiation factor 6 Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 108010003059 aggrecanase Proteins 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229940061720 alpha hydroxy acid Drugs 0.000 description 1
- 150000001280 alpha hydroxy acids Chemical class 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000006041 cell recruitment Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 108010053500 delicious peptide Proteins 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000004728 ear cartilage Anatomy 0.000 description 1
- 210000001162 elastic cartilage Anatomy 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002409 epiglottis Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000004349 growth plate Anatomy 0.000 description 1
- 108010006772 hemorrhagic metalloproteinase Proteins 0.000 description 1
- 210000003035 hyaline cartilage Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000002263 laryngeal cartilage Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004446 longitudinal ligament Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000005499 meniscus Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000002184 nasal cartilage Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000009818 osteogenic differentiation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920002463 poly(p-dioxanone) polymer Polymers 0.000 description 1
- 229920002627 poly(phosphazenes) Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000622 polydioxanone Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012256 transgenic experiment Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000002689 xenotransplantation Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Abstract
A process for inducing fibroblast to become cartilage cell includes culturing the fibroblasts in the culture liquid contaniing the cartilage form generating protein for 3-30 days to generate cartilage cells, and separating them for culture liquid.
Description
Technical field
The present invention relates to biology and organizational engineering field, relate more specifically to induce inoblast to form chondrocyte's method.
Background technology
Cartilaginous tissue mainly plays effects such as support, protection, dispersive stress as a kind of reticular tissue.Can be divided into hyaline cartilage (joint cartilage, costicartilage, nasal cartilages, tracheal cartilage), elastic cartilage (epiglottic cartilage, credulous bone), fibrous cartilage (meniscus) three classes on the histology.They all are the single organization of only containing the chondrocyte on histology, inner no blood vessel, and nutrition mainly relies on the infiltration of tissue juice.
On structure, the chondrocyte is wrapped in the cartilage cavities, and cartilage capsule thick around it is separated chondrocyte and surrounding tissue as one natural cover for defense.Therefore, cartilaginous tissue has a little less than the antigenicity, is difficult for being discerned and attacking by body immune system and has characteristics of light immunological rejection.Early stage result of study shows, the allogeneic chondrocyte can be survived in the acceptor body and be kept secreting the function of matrix.
It is one of clinical common disease that richets is decreased, as the various articular surface illness that caused by factors such as wound, infection, autoimmunization, tumours; Auricular cartilage, nose, laryngeal cartilage support damaged, lopsided; And the wound of bone in children epiphyseal growth plate, early close, necrosis etc.Because cartilaginous tissue does not almost have self-repairing capability, so must utilize cartilage or other equivalent material to repair.
From body and allosome cartilage transplantation is present common method.It is reliable to transplant curative effect from the body cartilaginous tissue, but must be cost to sacrifice human normal tissue, and the patient will suffer the misery of once performing the operation more.In addition, the autologous tissue source is limited, and the fritter graft also has yielding problem.Allosome and heterograft have immunogenicity again.
At present,, also exist the defective of aspects such as histocompatibility, material mechanical characteristic, still can not satisfy every requirement clinically though some artificial synthetic cartilaginous tissue surrogates are arranged.The synthetic equivalent material only possesses filling, supports and keeps effect attractive in appearance, and the abiology function still can not reach truly structure and reconstruction.
Organizational engineering is that new approach has been opened up in the damaged treatment of cartilaginous tissue.The used in tissue engineering small amounts of cells is inoculated on the Biodegradable material then at amplification in vitro, is built into cell-material composite, is used for transplanting repair deficiency.
One of essential condition that makes up the organization engineered cartilage tissue is to obtain a large amount of chondrocytes that function and vigor are arranged.In high large mammal even human body, also can utilize the tissue engineering technique regenerating cartilage tissue, but these are studied applied seed cell and are the chondrocyte, and can only be with former generation or the chondrocyte of going down to posterity in early days, and the chondrocyte after external a large amount of amplification is easily taken place aging and is dedifferented, and is difficult to form in vivo cartilaginous tissue.Very limited from body chondrocyte source, allogeneic and xenogenesis chondrocyte can't stop immunological rejection again, the chondrocyte though somatomedin can increase in a large number, but its expense costliness, still uncertainly, these all make cartilage tissue engineered being difficult at clinical application to institute's formative tissue long-term effect in vivo.
Cartilage tissue engineered seed cell should possess wide material sources, it is convenient and little to the body wound to draw materials, the in-vitro multiplication ability is difficult for characteristics such as aging by force.And mainly take from from the cartilage seed cell source of body cartilaginous tissue very limited at present, very easily dedifferente in chondrocyte's vitro culture process of end differentiation eventually, amplification quantity can not satisfy needs (the VonderMark K of tissue construction, Gauss V, Vonder Mark H, et al.Relationship between cell shape and type of collagen synthesized aschondrocytes lose their cartilage phenotype in culture.Nature, 1997,267:531-532).In conjunction with developmental biology research, widely distributed dermal fibroblast and chondrocyte derives from mesodermal mesenchyme cell (Mesenchymal cells) in the body, different with the chondrocyte is that inoblast has kept the strong biological characteristics of mesodermal mesenchyme ability of cell proliferation, repeatedly goes down to posterity still to keep multiplication capacity preferably.Studies show that, inoblast can be expressed special ground substance of bone (Inaba K in specific external environment, Matsunaga S, Ishidou Y, et al.Effect oftransforming growth factor-beta on fibroblasts in ossification of theposterior longitudinal ligament.In Vivo.1996 10:445-449.).Yet be not the report that seed cell makes the chondrocyte still up to the present, with inoblast.
In sum, seed cell source deficiency is the main difficulty of the cartilage tissue engineered clinical application of restriction, so this area presses for the seed cell of developing new preparation cartilage, and with the chondrocyte and the cartilage graft of its preparation.
Summary of the invention
Purpose of the present invention just provides a kind of seed cell of cartilage graft of wide material sources, and the method for preparing chondrocyte and graft with this seed cell.
In a first aspect of the present invention, a kind of chondrocyte's of preparation method is provided, comprise step:
(a) under the condition that is fit to growth, inoblast was cultivated 3-30 days in the inoblast nutrient solution that contains 10-1000 nanograms/milliliter CKMP (especially CDMP-1), formed the chondrocyte thereby inoblast is induced;
(b) from culture, isolate the chondrocyte.
In another preference, described growth conditions is 37 ± 2 ℃, CO
25 ± 3%.
In another preference, described nutrient solution is F-12, DMEM or the RPMI-1640 that contains 0-20% (more preferably 3-15%) foetal calf serum.
In another preference, described inoblast is people's a inoblast.
In another preference, the fibroblastic density described in the step (a) is 0.5 * 10
4-5 * 10
5Individual/ml.
In another preference, described inoblast is an inoblast former generation or that go down to posterity.
In a second aspect of the present invention, provide chondrocyte with method preparation of the present invention.
In a third aspect of the present invention, a kind of organization engineered cartilage graft is provided, it comprises:
(a) pharmaceutically acceptable Biodegradable material;
(b) with the chondrocyte of the method for the invention preparation, its content is 1 * 10
5-5 * 10
7Individual cells/ml, or 1 * 10
5-5 * 10
7Individual cell/cm
3
In another preference, described pharmaceutically acceptable Biodegradable material is selected from down group: poly(lactic acid), polyglycolic acid, poly(lactic acid)-polyglycolic acid multipolymer, Pluronics, polyhydroxybutyrate, poly-acid anhydrides, poly-phosphazo, polyamino acid, false polyamino acid, poe, polyester urethane, polycarbonate, polyoxyethylene glycol, polyethylene oxide, poly-P-Dioxane ketone, collagen, gelatin, ammonia polyose of candy, chitosan, chitin, alginates, calcium alginate gel, acellular matrix, and composition thereof.
In a fourth aspect of the present invention, a kind of fibroblastic purposes is provided, it is used to prepare the chondrocyte.
In a fifth aspect of the present invention, a kind of method for preparing the organization engineered cartilage graft is provided, it comprises step: the chondrocyte of the method for the invention preparation is mixed formative tissue through engineering approaches cartilage graft with pharmaceutically acceptable Biodegradable material.
Description of drawings
Fig. 1 has shown inoblast form phase microscope observations.
A) not inducing the group cellular form is spindle shape (* 100);
B) induce the group cellular form to change (shown in the arrow) to chondroblast sample polygon by spindle shape; The square frame inner cell is a vitro culture people s-generation normal cartilage cell (* 100).
Fig. 2 has shown that the cultured in monolayer in vitro inoblast induces group (A-C) and non-I, the II of group (D-F), the immunofluorescence detected result of III Collagen Type VI of inducing.Antibody is FITC mark (green fluorescence), and nucleus is that the PI lining dyes (red fluorescence).
A) CDMP-1 induces the visible obviously II Collagen Type VI of inoblast to express, and the frame inner cell is low power finding (* 100);
B) and C) inducing cell is still expressed I, III Collagen Type VI (* 100);
D) vitro culture normal fibroblast II Collagen Type VI immunofluorescence feminine gender; E, F) normal fibroblast I, III Collagen Type VI are expressed positive.
Fig. 3 has shown the detected result of centrifuge tube being cultivated the culturing cell agglomerate.
A) Alicain Blue dyeing, the cell mass Celeste is dyed, and shows GAG secretion (* 100).
B) Masson ' s Trichrome dyeing, collagen stroma indigo plant is dyed (* 200)
Fig. 4 has shown centrifuge tube method culturing cell agglomerate II Collagen Type VI immunohistochemical staining (ABC method) detected result.
A) visible a large amount of II Collagen Type VI depositions (* 100) in cell and the matrix.
B) the local interior brown yellow granule (* 200) of back visible cell that amplifies)
Fig. 5 has shown the RT-PCR detected result to inducing cell.
A) induce group cell RT-PCR to detect Aggrecan, II collagen mRNA band of expression, do not induce the group result negative.
B), C) inducing cell RT-PCR detects and expresses I, III Collagen Type VI.
Fig. 6 has shown that the Western blotting detects the result that the II Collagen Type VI is expressed.The experimental group inoblast induces back II collagen type to be expressed as the positive, same band occurs with the normal cartilage cell; Control group is fibroblastic does not see II collagen type band of expression.
Embodiment
The inventor is through extensive and deep discovering, CKMP (cartilage-derived morphorgentic protein, CDMP) be particularly suitable as and induce inoblast to be differentiated to form chondrocyte's inducible factor, can induce inoblast effectively the body profile chondroblast.Finished the present invention on this basis.
Term
Term " purifying or isolating " refers to purifying or isolating material is substantially devoid of other cells, protein or polypeptide, for example cytokine of purifying, or isolating chondrocyte.
Term " xenotransplantation " refers to required living organisms material (as inoblast) is taken out and is applied to the method for another species object from a certain species.
Term " autotransplantation " refers to required living organisms material (as inoblast) is taken out and is applied to same patient's method from certain patient.
Term " heteroplastic transplantation " refers to required living organisms material (as the chondrocyte) is taken out the method that also is applied to again with another Different Individual of species from certain individuality.
Inoblast
Fibroblastic source is not particularly limited among the present invention, and a kind of preferred source is from the corium from body.Usually, inoblast of the present invention is from from body or allogeneic.Inoblast can be an inoblast former generation or that go down to posterity.
It all is as known in the art separating and cultivating the fibroblastic method of acquisition.A kind of preferable methods is to get the dermis of skin tissue, shred and move in the centrifuge tube, add about 10 times to 0.1% type i collagen enzymic digestion of tissue volume, 37 ℃, 2 hours, it is centrifugal that 100 order cellular filters filter the back, 1000 changeed 5 minutes, supernatant discarded adds and contains 10% FBS DMEM nutrient solution mixing cell, with 2 * 10
4/ cm
2Be inoculated in the 100mm culture dish and cultivate, when treating cytogamy to 80%-90%, go down to posterity, the density that goes down to posterity is 1 * 10
4/ cm
2Preferred the 2nd～18 generation cell be used to prepare artificial cartilage.
The preferred inoblast of one class is the s-generation～hexabasic inoblast of vitro culture.The potential that the inoblast of this moment has stronger multiplication capacity and breaks up to the chondrocyte.
Inducible factor
As used herein, " inducible factor " refer to CKMP (Cartilage-DerivedMorphogenetic Proteins, CDMPs).This is the special somatomedin of a class in the BMP family, and it brings into play important regulation in the cartilage development process, and wherein CKMP-1 (CDMP1) and CKMP-2 (CDMP2) are preferred.More preferably be CDMP1.
As used herein, " cell induction liquid " refers to contain the inoblast nutrient solution of 10-1000 nanograms/milliliter (preferably 20-500ng/ml, more preferably 50-200ng/ml) CKMP.
Except CDMP, but other factors of coupling also in the method for the invention, for example transforming growth factor-beta (transforming growth factor beta, TGF β) 1,2,3, Delicious peptide (bonemorphorgenetic protein, BMP) 2,6,7.Other induction factors of coupling cultivate, in specific cartilage cell epimatrix composition, cultivate, use retardance as: low-oxygen environment or activate specific cell signaling path, with chondrocyte's co-cultivation etc.
Induce inoblast to form the chondrocyte
Inducing step is the key of the inventive method.Usually, under the condition that is fit to growth, inoblast is being added 10-1000 nanograms/milliliter (20-500ng/ml preferably, 50-200ng/ml more preferably) cultivates 3-30 days (preferably 5-15 days) in the inoblast nutrient solution of CKMP, just can make inoblast induce the formation chondrocyte.
A kind of preferable methods is that single-layer culturing cell is induced, and carries out cell induction in the CDMP inoblast substratum (as the F-12 substratum) that contains about 10%FBS and about 10-1000ng/ml, changes nutrient solution one time in per three days, induces 3-30 days.
Another kind of preferred method is that the centrifuge tube culturing cell is induced.Inoblast is left the heart about 1500, abandon and add cell induction liquid behind the supernatant, cell mass has been shaken, be suspended in the nutrient solution, induced 3-30 days.
Through behind the inducing culture, can pass through Masson ' s Trichrome, Alcian Blue and immunohistochemical staining, RT-PCR, Western engram analysis etc. carry out qualitative and sxemiquantitative is identified, isolate the chondrocyte then from culture.
Biodegradable material
The material that can be used for organization engineered cartilage of the present invention is medically acceptable Biodegradable material, comprises (but being not limited to):
(a) degradability synthesized polymer material, for example poly-alpha hydroxy acid (as polylactic acid PLA, polyglycolic acid PGA, polyhydroxybutyrate PHB etc.), poly-acid anhydrides (polyanhydrides), poly-phosphazo (polyphosphazenes), polyamino acid (polyamino acid), false polyamino acid (pesudo-polyamino acid), poe (polyorthoesters), polyester urethane (polyesterurethane), polycarbonate (polycarbonate), polyoxyethylene glycol, poly-P-Dioxane ketone (polydioxanone) etc.;
(b) natural degradable material, for example collagen (collagen), gelatin (gelatin), ammonia polyose of candy (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginates, calcium alginate gel etc.; Various acellular matrixes;
(c) syringeability material is as Pluronic (a kind of commercially available poly-epoxy second propylene commodity), calcium alginate gel etc.;
The matrix material of (d) matrix material of the mixture of above-mentioned materials or matrix material or multipolymer, especially macromolecular material and natural materials, and solid material and syringeability material.Multipolymer: polyglycolic acid-copolymer of poly lactic acid (lactic-co-glycolic acid, PLGA)
Preferred medically acceptable Biodegradable material is the material of syringeability, for example calcium alginate gel, Pluronic gel etc.
The cell concn of chondrocyte in the organization engineered cartilage of the present invention is about 2 * 10 usually
7/ ml to 5 * 10
7/ ml or higher.When material is the syringeability material, adjust chondrocyte's concentration with this fluent material; When material is the solidity material, adjust chondrocyte's concentration with nutrient solution, with the solidity material mixing, the ratio of nutrient solution and solidity material is not particularly limited when wherein mixing then, but with this solid material can adsorb nutrient solution maximum be as the criterion.
In organization engineered cartilage graft of the present invention, also can add or compound other various cytokines, somatomedin, various transgene component, thereby keep chondrocyte's phenotype, promote chondrocyte's growth or matrix synthesis capability etc.
The organization engineered cartilage graft
Chondrocyte with the inventive method preparation can be used for making up the organization engineered cartilage graft, and the described chondrocyte who is about to some amount mixes with pharmaceutically acceptable Biodegradable material and gets final product.
Organization engineered cartilage graft with the inventive method forms is divided into two classes, the class mixture that to be the chondrocyte constitute with the syringeability biomaterial, interior each positions of body such as that this mixture can be injected directly into is subcutaneous, cartilage defect place.Another kind of is the mixture that chondrocyte and non-syringeability biomaterial constitute, each position in the bodies such as that this mixture can directly implant is subcutaneous, cartilage defect place.
Biodegradable material is solid-state in a preference.At this moment, to make concentration be about 5 * 10 for available above-mentioned cell and nutrient solution
7The cell suspension of/ml forms mixture with biomaterial polyglycolic acid/polylactic acid bracket (PGA/PLA) then, and this mixture size, shape are determined according to the size and the shape of cartilage defect.One thoughtful three weeks of vitro culture, changed liquid once in per two to three days, to guarantee cytotrophy, each position in the bodies such as subcutaneous, the cartilage defect place of when vitro tissue through engineering approaches cartilage begins to take shape, implanting.
In another preference, Biodegradable material is syringeability material (Pluronic gel, a calcium alginate gel etc.).Adjusting cell concn with this fluent material is about 5 * 10
7/ ml is directly used in damaged reparation in subcutaneous injection or the body.
Application method
When Biodegradable material was the syringeability material, cell and biomaterial composites can directly be implanted subcutaneous or the interior cartilage defect place of body.When Biodegradable material when being solid-state, cell and biomaterial be external compound, in bio-reactor, form the organization engineered cartilage that is fit to various cartilage defects sizes and shape after, cartilage defect place again implants.
Major advantage of the present invention is:
(1) uses autogenous cell, and once draw materials and both can obtain enough cell concentrations;
(2) fibroblastic acquisition operations is simple, and is minimum to patient trauma, need not to be in hospital, and can repeat to draw materials;
(3) extracorporeal culturing method is easy to learn, and is easy to utilize.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Inoblast is cultivated and goes down to posterity
Get the dermis of skin tissue that excises in the posthetomy under the aseptic condition, shred and move in the centrifuge tube, add about 10 times to 0.1% type i collagen enzyme (WORTHINGTON company) digestion of tissue volume, 37 ℃, 2 hours, it is centrifugal that 100 order cellular filters filter the back, 1000 changeed 5 minutes, supernatant discarded adds and contains 10% FBS DMEM nutrient solution (GIBCO) mixing cell, with 2 * 10
4/ cm
2Be inoculated in the 100mm culture dish (FALCON, FranklinLakes NJ) and cultivate, when treating cytogamy to 80%-90%, go down to posterity, the density that goes down to posterity is 1 * 10
4/ cm
2Amplification in vitro is after the s-generation, and grouping experimentizes.
Embodiment 2
The single-layer culturing cell revulsion is induced and is formed the chondrocyte
In the present embodiment, induce the formation chondrocyte with the single-layer culturing cell revulsion.Method is as follows:
To 100 μ g/ml, adding F-12 (Gibco company) nutrient solution that contains 10%FBS, to make its final concentration be 100ng/ml, is mixed with cell induction liquid with 0.1%BSA dilution CDMP1 somatomedin (Research Diagnostics).Got the s-generation inoblast inoculation 6-12 hour, cell induction changed nutrient solution one time in per three days, induce 7 days (at 37 ℃, 5%CO
2, saturated humidity), separate obtaining the chondrocyte.
Embodiment 3
The centrifuge tube culturing cell is induced
In the present embodiment, induce the formation chondrocyte with the single-layer culturing cell revulsion.Method is as follows:
The induced liquid preparation is with embodiment 2.Collect the inoblast 1500 that was passaged to for the 6th generation and left the heart 5 minutes, abandon and add cell induction liquid behind the supernatant, cell mass has been shaken, induced acquisition chondrocyte group 7 days.
Cut into slices behind the paraffin embedding, for Masson ' s Trichrome, Alcian Blue and immunohistochemical staining etc. are identified and are used.
Embodiment 4
Induce the detection of effect
One, detection method
(a) the cell laser co-focusing detects
With control group among the embodiment 2 and experimental group cell respectively after cultivating and inducing 7 days, 95% acetone fixed 10 minutes, the PBS rinsing, 10% sheep blood serum sealing 30 minutes, the anti-I of rabbit, II, III Collagen Type VI antibody (1:100, Chemicon company) hatches 1 hour respectively, the PBS rinsing, (the 1:1000 dilution of FITC goat anti-rabbit igg, Oncogene company) hatches 40 minutes, PBS rinsing, PI (Propidiumiodide propidium iodide) (1:1000 dilution, Sigma company) incubated at room is 10 minutes, mounting after the PBS rinsing.Sample behind the mark is placed two-photon laser confocal microscope (available from Zeiss company, model LSN510), laser excitation wavelength 488nm, mirror is observed down.The gained image carries camera system input computer by mirror preserves automatic camera.
(b) cell mass II Collagen Type VI immunohistochemistry detects
After the section dewaxing entry with preparation among the embodiment 3, PBS rinsing, 3%H
2O
2The sealing endogenous peroxydase drips 0.4% stomach en-and hatches 30min for 37 ℃, adds rabbit anti-people II Collagen Type VI monoclonal antibody (1:100, Chemicon company), put wet box and spend the night for 4 ℃, drip goat antirabbit two anti-(1:100, DAKO), hatch 30min for 37 ℃, after the PBS rinsing, the DAB colour developing.Hematorylin is redyed, the resinene mounting.Not add a resisting sample as blank.
(c) cell collagen is expressed the RT-PCR detection
Collect respectively control group and experimental group cultivate and induce 7 days after cell, detect II Collagen Type VI and aggrecan (Aggrecan) expression.Trizol (Invitrogen company) extracts total mRNA, and the mRNA that detects I, II Collagen Type VI and aggrecan expresses.According to RT-PCR test kit (Takara) operation instructions, carry out reverse transcription and pcr amplification reaction.Corresponding primer (synthetic by Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) sequence sees Table 1.Reaction conditions: 94 ℃ of sex change, 55 ℃ of annealing, 35 circulations.
Table 1. primer sequence and amplified production size
| Upstream primer | Downstream primer | The product size | |
| Aggrecan | 5′-atgcccaagactaccagtgg | 5′-tcctggaagctcttc?tcagt | 510bp |
| COL-I | 5′-cttggtctcgtcacagatca | 5′-tgttcagctttgtggacctc | 210bp |
| CoLII | 5′-cttgggcacctcgggctcctttag | 5′-tccccggcactcctggcactgat | 510bp |
| COL-III | 5’-tggacgaaatggagaa | 5’-cgcctttaccaccaggacta | 370bp |
(d) cell II Collagen Type VI secretion Western trace detects
Collect respectively control group and experimental group cultivate and induce 7 days after cell, total cellular score is equal 1 * 10
6Individual, add three decontamination lysate 1ml, move into behind the lysing cell in the 1.5ml Eppendorf tube, 10000rpm, 4 ℃ are centrifugal 5 minutes.Get supernatant, add equal-volume 2 * SDS Loading buffer, beta-mercaptoethanol 2 μ l/100 μ l.100 ℃ of boiling water sex change 10 minutes in centrifugal 2 seconds in wink, are waited to be chilled to room temperature and are promptly gone up sample.Polyacrylamide gel electrophoresis 10mA electrophoresis 3-4 hour.After electrophoresis finishes, 90v, 4 ℃ were changeed film 2 hours, and 4 ℃ of sealings of sealing damping fluid (PIERCE company) are spent the night.37 ℃ add II Collagen Type VI mouse-anti one anti-(1:500 successively, Sigma company), biotinylation two anti-(1:500, Vector Lab company), streptavidin-AP (1:3400, Pierce company) hybridizes, after hybridization is amplified step by step, drip 1-Step NBT/BCIP (Pierce company) colour developing liquid, carry out interpretation of result after the colour developing.
Two, result
Cellular form changes: after application CDMP1 induced 3 days, the inoblast form is not seen did not have obviously change; When inducing after 7 days, the inoblast of visible spindle shape begins to change to polygon, Polygons, and is closely similar with chondrocyte's form.Cellular control unit does not see that obvious form changes (Fig. 1).
Cell collagen is expressed and observed: laser confocal microscope is observed and is found that the normal fibroblast of monolayer culture (not inducing group) is I, III Collagen Type VI luciferase expression only, does not see II Collagen Type VI fluorescencepositive cell.After application CDMP induces, II Collagen Type VI fluorescencepositive cell showed increased, but observation of cell is still expressed I, the III Collagen Type VI fluorescence positive simultaneously, and fluorescence intensity slightly weakens (Fig. 2).
Cell mass induces that back matrix is synthetic to be detected: use centrifuge tube method cultured cells agglomerate, after using CDMP1 and inducing 7 days, the Masson ' s Trichrome visible matrix that dyes is the diffusivity bright green, shows that collagen synthesizes vigorous (Fig. 3 A).The Alcian Blue visible matrix that dyes is homogeneity Celeste indigo plant and dyes, and shows that GAG deposits (Fig. 3 B) in a large number.Cell mass II Collagen Type VI immunohistochemical staining finds, brown xanchromatic size distribution in cell with extracellular matrix in; The II Collagen Type VI is expressed in that cell density is higher, the zone of matrix synthesis of densified dyeing strong (Fig. 4).Control group detects negative.
II Collagen Type VI and Aggrecan express RT-PCR and detect: the inoblast CDMP1 of monolayer culture induced back 7 days, and RT-PCR detects size and is to conform to the II Collagen Type VI of 510bp and Aggrecan amplified production with positive control chondrocyte amplification.Do not see amplification positive band (Fig. 5 A) for inducing group.Induce inoblast still can detect I, III collagen mRNA express (Fig. 5 B, C).
The II Collagen Type VI is expressed the Western engram analysis and detected: the inoblast CDMP1 of monolayer culture induced back 7 days, can detect the II collagen type and express, and is identical with positive controls chondrocytes expressed band.Do not induce the control group inoblast not see II collagen type (Fig. 6).
Embodiment 5
The preparation of tissue engineering bone/cartilage graft
With the chondrocyte of preparation in embodiment 2 or 3, respectively with 5 * 10
7The final concentration of individual cell/ml mixes, thereby makes the tissue engineering bone/cartilage graft with 30%Pluronic F-127 (a kind of commercially available poly-epoxy second propylene commodity)/DMEM.
Discuss
As the class somatomedin in the TGF-beta superfamily, people CDMP is at first gone out by clone and separate in the joint cartilage, mainly comprises two hypotypes of CDMP1 and CDMP2, BMP14 and BMP13 in the corresponding BMP of the difference family; Growth and differentiation factor in corresponding mouse BMP of the while family (Growth and Differentiation Factor, GDF).
In the whole family members of the present BMP that finds, CDMP is a most special class somatomedin relevant with growth with the generation of cartilage form, brings into play important regulation in graphic formation (patterning), cartilage generation and the long bone process of growth of bones of limbs.
Discover, CDMP1 specific expressed in embryo development procedure joint and joint cartilage form position (TsumakiN, Tanaka K, Arikawa-Hirasawa E, et al.Role of CDMP-1 inskeletal morphogenesis:Promotion of mesenchymal cell recruitment andchondrocyte differentiation.J.Cell Biol., 1999.144:161-173); Development model (Storm EE and Kingsley DM.GDF5 coordinates bone and jointformation during digit development.Dev.Bio.1999 in the body, 209:11-27) and transgenic experiments all confirm, CDMP1 is mainly by promoting the gathering (consendation) of mesenchymal cell, improve the quantity of local cartilage precursor cell, quicken joint cartilage formation, chondrocyte's differentiation and regulate the growth of bone tissue.All can detect the expression of CDMP in the birth back and the articular cartilage tissue of growing up, prompting CDMP can continue to keep the growth of normal articular cartilage after birth; External organ culture is discovered, there are a large amount of CDMP to express in the joint cartilage of damage, under the effect of the exogenous CDMP factor, cartilage matrix is synthetic obviously to be increased, prompting CDMP can promote injury repairing process (the Ludwig E of joint cartilage, Chee KNG, Robert U, et al.Presence of cartelage-derived morphogenetic proteins in articularcartelage and enhancement of matrix replacement in vitro.Arthriti.Rheumat.1998,41:263-273).Above-mentioned studies show that, CDMP is mainly by the differentiation of matter precursor cell between regulating, and the almost all biological that has participated in cartilaginous tissue generation, growth and injury repairing is learned process, is the somatomedin that important adjusting chondrocyte breaks up.
It is generally acknowledged that dermal fibroblast is to break up whole completely opisthosoma cell, though draw materials and cultivate simple, the amplification in vitro ability is strong, no longer possesses the ability to the other types cytodifferentiation of stem-like cell, only originate as dermal tissue engineering seed cell.Yet, use the CDMP1 somatomedin among the present invention, act on the dermal fibroblast of vitro culture, but but find cell expression specificity chondrocyte matrix after 7 days: II Collagen Type VI and aggrecan, chondrocyte's sample with cellular form changes simultaneously, and this has confirmed that the diploid fibroblast of whole end differentiation still has the potentiality as the seed cell source that makes up its hetero-organization.
Cartilage cell epimatrix mainly is made up of II type collagen fiber (COLII) and proteoglycan, and wherein the main component of proteoglycan is an aggrecan.Collegen filament form network structure and have very high tensile strength, the hairbrush spline structure that is present in the aggrecan molecule in the network can make its better counter pressure support the structure of cartilaginous tissue jointly with the II Collagen Type VI, keeping the biological property of cartilaginous tissue, therefore the expression of COLII and aggrecan is the most special mark of chondrocyte (Micky D.Tortorella at present, Michael A., et al.The Interglobular Domain of CartilageAggrecan Is Cleaved by Hemorrhagic Metalloproteinase HT-d (AtrolysinC) at the Matrix Metalloproteinase and Aggrecanase Sites.J.Bio.Chem.1998,273:5846-5850).Dermal fibroblast itself is not expressed COLII and aggrecan, under culture system effect of the present invention, the inoblast of monolayer culture is after CDMP induces 7 days, cellular form has taken place by the transformation of fusiformis to chondrocyte's sample polygon form, and immunofluorescence, Western-Blot and RT-PCR have all confirmed the expression of II collagen; RT-PCR result also observes inoblast and induces the back to express aggrecan.It is mesenchymal precursor cells gathering in the simulation cartilage form generating process, atomization that centrifuge tube is cultivated, and the research stem cell is to the extracorporeal culturing method of chondrocyte's differentiation.In order further to confirm that above-mentioned result of study, the inventor adopt centrifuge tube culture technique research inoblast to induce atomization under state of aggregation.Discover that under the CDMP1 effect, the cell mass of inducing 7 days confirms to have a large amount of acidic mucopolysaccharide depositions through A Lixinlan dyeing, the synthetic of collagen and mucopolysaccharide also observed in Masson ' s dyeing.Immunohistochemical methods, Western-Blot and RT-PCR all detect the expression of COLII and aggrecan behind the centrifuge tube inducing culture.Above-mentioned result of study and bone marrow stroma stem cell are induced the report of differentiation (the Johnstone B that conforms to the chondrocyte, Hering TM, Caplan AI, et al.In vitro chondrogenesisof bone marrow derived mesenchymal progenitor cells.Exp cell res, 1998,238:265-272.).
Find in this research that though inoblast is expressed COLII and aggrecan under the CDMP1 effect, the expression of I, III Collagen Type VI does not disappear, show that the chondrocyte through inductive inoblast and nature also has a certain distance.Lennon (Lennon D, Haynesworth SE, ArmDM, et al.Dilution of human mesenchymal stem cells with dermal fibroblasts andthe effects in vitro and in vivo osteochondrogenesis.Dev.Dyn.2000,219:50-62) wait human dermis inoblast and bone marrow stroma stem cell mixed with 25-50%, still visible cartilaginous tissue formed after centrifuge tube was cultivated and induced, and had significantly reduced the consumption of bone marrow stroma stem cell.The inventor thinks, by inducing of CDMP, might further reduce the ratio of bone marrow stroma stem cell in the cartilaginous tissue building process.Simultaneously because cartilage forms and often is the commitment in the os endochondrale process, so the inventor thinks inoblast expression specificity cartilage matrix among the present invention, can not get rid of is the stage of inoblast to the skeletonization phenotypic differentiation.Find (GruberR inducing in the differentiation research of marrow stromal cell, Mayer C, Schulz W, et al.St imulatory effects ofcarti lage-derived morphogenetic proteins 1 and 2 on osteogenicdifferentiation of bone marrow stromal cells.Cytokine.2000,12:1630-1638), CDMP1,2 can promote that all bone marrow stroma stem cell breaks up to osteoblasts in vitro, but its osteogenesis is starkly lower than BMP-6 and OP-1; Though there is the IIA procollagen type to express, do not detect as the IIB procollagen type of ripe chondrocyte's matrix and secrete with aggrecan, somatomedin action time only is 4 days in this research.In another research, when the CDMP induction time extended to 7 days, periosteum source sexual cell had showed skeletonization and the multidirectional differentiation that becomes cartilage, and visible obviously II Collagen Type VI and aggrecan are expressed, and become the cartilage effect obviously to be better than osteogenesis.Therefore, CDMP is necessary to continue the function of its skeletonization of observation or one-tenth cartilage under different action times, show skeletonization and the dual intensity effect that becomes cartilage at different cells under same action condition.
Of the present invention having experimental results show that, dermal fibroblast is induced by CDMP-1, under monolayer culture and centrifuge tube culture condition, the phenomenon to chondrocyte's phenotypic differentiation has all appearred, and inoblast can be synthesized specificity chondrocyte matrix: II procollagen type and aggrecan.This result of study has not yet to see bibliographical information, and prompting is by the inducing action of CDMP, and inoblast has the possibility of originating as cartilage tissue engineered new seed cell.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (7)
1. a method for preparing the chondrocyte is characterized in that, comprises step:
(a) under the condition that is fit to growth, inoblast was cultivated 5-15 days in the inoblast nutrient solution that contains 10-1000 nanograms/milliliter CKMP, formed the chondrocyte thereby inoblast is induced;
(b) from culture, isolate the chondrocyte.
2. the method for claim 1 is characterized in that, described growth conditions is 37 ± 2 ℃, CO
25 ± 3%.
3. the method for claim 1 is characterized in that, described nutrient solution is the F-12 that contains the 0-20% foetal calf serum, DMEM or RPMI-1640.
4. the method for claim 1 is characterized in that, described inoblast is people's a inoblast.
5. the method for claim 1 is characterized in that, the fibroblastic density described in the step (a) is 0.5 * 10
4-5 * 10
5Individual/ml.
6. the method for claim 1 is characterized in that, described inoblast is an inoblast former generation or that go down to posterity.
7. a method for preparing cartilage graft is characterized in that, comprises step:
(a) under the condition that is fit to growth, inoblast was cultivated 5-15 days in the inoblast nutrient solution that contains 10-1000 nanograms/milliliter CKMP, formed the chondrocyte thereby inoblast is induced;
(b) from culture, isolate the chondrocyte;
(c) chondrocyte who obtains in the step (b) is mixed with pharmaceutically acceptable Biodegradable material, form cartilage graft, chondrocyte's content described in the wherein said graft is 1 * 10
5-5 * 10
7Individual cells/ml, or 1 * 10
5-5 * 10
7Individual cell/cm
3
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100165142A CN100482784C (en) | 2004-02-25 | 2004-02-25 | Method for inducing fibroblast to form cartilage cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100165142A CN100482784C (en) | 2004-02-25 | 2004-02-25 | Method for inducing fibroblast to form cartilage cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1661010A CN1661010A (en) | 2005-08-31 |
| CN100482784C true CN100482784C (en) | 2009-04-29 |
Family
ID=35010562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100165142A Expired - Lifetime CN100482784C (en) | 2004-02-25 | 2004-02-25 | Method for inducing fibroblast to form cartilage cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100482784C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748095B (en) * | 2008-11-28 | 2013-05-08 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| EP3417888A1 (en) * | 2017-06-25 | 2018-12-26 | co.don AG | Method for producing transplantable cartilage tissue |
| CN107267448A (en) * | 2017-07-28 | 2017-10-20 | 陈强 | A kind of induced fibroblast is the method for in vivo bioreactor |
-
2004
- 2004-02-25 CN CNB2004100165142A patent/CN100482784C/en not_active Expired - Lifetime
Non-Patent Citations (4)
| Title |
|---|
| 骨髓间干细胞软骨细胞表型定向诱导分化的实验研究. 中华整形外科杂志，第18卷第1期. 2002 |
| 骨髓间干细胞软骨细胞表型定向诱导分化的实验研究. 中国运动学杂志，第23卷第1期. 2004 |
| 骨髓间干细胞软骨细胞表型定向诱导分化的实验研究. 中华整形外科杂志，第18卷第1期. 2002 * |
| 骨髓间干细胞软骨细胞表型定向诱导分化的实验研究. 中国运动学杂志，第23卷第1期. 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1661010A (en) | 2005-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101589139B (en) | Artificial cartilage containing chondrocytes obtained from costal cartilage and preparation process thereof | |
| Man et al. | Transplantation of allogenic chondrocytes with chitosan hydrogel-demineralized bone matrix hybrid scaffold to repair rabbit cartilage injury | |
| Mitra et al. | Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells | |
| Trojani et al. | Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells | |
| Choi et al. | The chondrogenic differentiation of mesenchymal stem cells on an extracellular matrix scaffold derived from porcine chondrocytes | |
| Chen et al. | In vivo tendon engineering with skeletal muscle derived cells in a mouse model | |
| Deponti et al. | Collagen scaffold for cartilage tissue engineering: the benefit of fibrin glue and the proper culture time in an infant cartilage model | |
| CN111197024B (en) | Pancreatic-like structure, construction method and application thereof | |
| Guasti et al. | Chondrogenic differentiation of adipose tissue-derived stem cells within nanocaged POSS-PCU scaffolds: a new tool for nanomedicine | |
| Breidenbach et al. | Fibrin gels exhibit improved biological, structural, and mechanical properties compared with collagen gels in cell-based tendon tissue-engineered constructs | |
| Peister et al. | Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds | |
| Hou et al. | Printing 3D vagina tissue analogues with vagina decellularized extracellular matrix bioink | |
| CN101084026A (en) | Poly (ethylene glycol) - diacrylate (pegda) - crosslinked comprising adipogenic mesenchymal stem cells | |
| Ma et al. | The graft of autologous adipose-derived stem cells in the corneal stromal after mechanic damage | |
| US20100197020A1 (en) | Tissue engineering tendon and construction methods in vitro thereof | |
| Levorson et al. | Cell-derived polymer/extracellular matrix composite scaffolds for cartilage regeneration, Part 1: investigation of cocultures and seeding densities for improved extracellular matrix deposition | |
| Dai et al. | Generation of rhBMP-2-induced juvenile ossicles in aged mice | |
| CN109847098B (en) | Composite biological scaffold material for repairing bone defect | |
| Yang et al. | Effects of scaffold geometry on chondrogenic differentiation of adipose-derived stem cells | |
| Teng et al. | Extracellular matrix powder from cultured cartilage-like tissue as cell carrier for cartilage repair | |
| Muiños-López et al. | Human amniotic mesenchymal stromal cells as favorable source for cartilage repair | |
| Chen et al. | Chm-1 gene-modified bone marrow mesenchymal stem cells maintain the chondrogenic phenotype of tissue-engineered cartilage | |
| Hou et al. | Dominant role of in situ native cartilage niche for determining the cartilage type regenerated by BMSCs | |
| Hagmeijer et al. | Meniscus regeneration combining meniscus and mesenchymal stromal cells in a degradable meniscus implant: An in vitro study | |
| Ghanavati et al. | Characterization of a three-dimensional organotypic co-culture skin model for epidermal differentiation of rat adipose-derived stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20090429 |