| Chapterâ… The isolation, culture and identificationadipose-derived mesenchymal stem cellsObjective To master the method of isolation and culture ofadipose-derived mesenchymal stem cells, and evaluate their growth andproliferation, still identify their biological characteristics.Methods The rat's inguinal and testicle fat pads were excised, thendigested with 0.075% collagenase, then the cells were cultrued inDulbecco modified Eagle medium (DMEM) with 10% new-born calfserum. The culture medium is replaced constantly in order tohomogenizing adipose-derived mesenchymal stem cells. Themorphology of ADMSCs was observed with inserted microscopeconstantly. Growth curve of the 3rd, 5th and 10th passage was made bymeans of MTT. The CD29, CD34 and CD44 expression of the 5th passagecells were detected by flow cytometry.Results A few of adherent cells may be observed at 24 hours later.The primary ADMSCs indicated long or short spindle-shape ormultangular shape at 48 hours later. Fibroblast-like cells manifoldprogressively after the 3rd day.Logarithm growth period emergences afterthe 5th day. Cells confluence more than 90% after the 8th day. Passagecells began to adhere within 2 hours after sub-cultrue, proliferaterapidly, most fibroblast-like. Passage cells confluence after the 6th day.The shapes of cells are homogeneous after the 3rd passage.Broadbrimmed-like cells emergence after the 10th passage. The Growthcurve shows that proliferation ability of the 3rd passage and 5th passagecells is more powerful than the 10th passage cells. Flow cytometry showsthat 95% and 96% of the 5th passage cells expressed CD44 and CD29respectively, while only 5% of the cells expressed CD34.Conclusion The method of isolation and culture of ADMSCs issimple and feasible. ADMSCs can be cultured in a long period and growstably, proliferate rapidly, may be used as a kind of seed cells for tissueengineering. Chapterâ…¡Chondrogenic potential of adipose -derivedmesenchymal stem cells in vitroObjective To explore chondrogenic potential of adipose -derivedmesenchymal stem cells cultured with a specific medium in vitro.Methods (1) Adipose -derived mesenchymal stem cells wereisolated and cultured according to carpterâ… . (2) The 5th cells werecollected and regulated into a concentration of 1×1010/L, still induced ina "micromass" with a specific Dulbecco medium containing new-borncalf serum of 0.01 volume fraction, tansforming growth factor-β110ug/L, insulin 6.25mg/L, transferrin 6.25mg/L, Dexamethasone1×10-7mol/L, and ascorbic acid-2-phosphate 50mg/L. (3)Chondrogenesis was assessed using Alcian blue staining, Toluidine bluestaining, Safranin O/Fast Green staining at 7 and 14 days, still collagenâ…¡immunohistochemistry and aggrecan immunofluorescence at 4, 7 and14 days after initial chondrogenic induction of adipose -derivedmesenchymal stem cells. (4) The mRNA expressions of Col2al, Agc1and Sox9 were detected by RT-PCR at 0, 14, 28 days after initialchondrogenic induction of adipose -derived mesenchymal stem cells. (5)The protein expressions of Col2al and Agc1 were detected byWestern-blot at 0, 14, 28 days after initial chondrogenic induction ofadipose -derived mesenchymal stem cells.Results (1) High-density micromass cultures of Adipose -derivedmesenchymal stem cells could assemble themselves into small translucentspheroids that were visible to naked eyes as early as 48 hours after initialchondrogenic induction. Over the course of 12 days, the volume ofcartilaginous nodule increased. The induced ADSCs indicated round,triangular or multangular shape. (2) The extracellular matrix wasstained positively for Alcian blue, Toluidine blue and Safranin O / FastGreen at 7 and 14 days after initial chondrogenic induction of adipose -derived mesenchymal stem cells, and indicated collagenâ…¡and aggrecanwere expressed positively. (3) RT-PCR indicated that high-densitymicromass cultures of Adipose -derived mesenchymal stem cells couldexpress Col2al mRNA, aggrecan mRNA and Sox9 mRNA at 2 and 4weeks after initial chondrogenic induction of adipose -derivedmesenchymal stem cells, but no express at 0 week groups. (4)Western-blot indicated that high-density micromass cultures of Adipose-derived mesenchymal stem cells could express the proteins of Col2aland aggrecan at 2 and 4 weeks distinctly after initial chondrogenicinduction of adipose -derived mesenchymal stem cells, only very weak at0 week groups.Conclusion Adipose -derived mesenchymal stem cells withhigh-density micromass cultures can differentiate into chondrocytes, stillexpress chondrocyte phenotype steadily under the specific inducingenvironment in vitro.Chapterâ…¢Chondrogenic differentiation of adipose-derivedmesenchymal stem cells in PLGA scaffoldsObjective To explore chondrogenic differentiation capability ofadipose-derived mesenchymal stem cells cultured and induced in PLGAscaffolds, and the feasibility of PLGA scaffolds as a carrier for it.Methods (1) PLGA copolymer scaffolds were sterilized by 70%ethanol and ultraviolet radiation in turn, dried in preparation. (2)Adipose -derived mesenchymal stem cells were isolated and culturedaccording to carpterâ… . The 5th passage cells were collected and regulatedinto a concentration of 4×1010/L, still inoculated into PLGA, followedby the specific induction medium containing new-born calf serum of 0.01volume fraction, tansforming growth factor-β1 10ug/L, insulin6.25mg/L, transferrin 6.25mg/L, Dexamethasone 1×10-7mol/L, andascorbic acid-2-phosphate 50mg/L. Noninduction groups was set. (3) The complex of cells and scoffolds was terminated in culture medium,embeded in paraffin and cut in section, still assessed using H-Estaining, Safranin O / Fast Green staining and Masson staining, stillcollagenâ…¡immunohistochemistry and aggrecan immunofluorescenceat 3 weeks after initial chondrogenic induction of the complex. Theimpanted cells and scaffolds were observed with scanning electronmicroscope. (4) The mRNA expressions of Col2al, Agc1 and Sox9were detected by RT-PCR at 3 weeks after initial chondrogenic inductionof adipose -derived mesenchymal stem cells cultured in PLGA scaffolds.Results (1) The complex of cells and scoffolds suspended in themedium at first, sunk at the bottom of medium after 2 days. Some cellsmay overglow scoffolds, but the tenacity was increased and the surfacewas lenitive in the induced groups. (2) Scanning electron microscopeshowed many spherical cells distributed in pores and on the surface of thescaffolds. Much more extracellular matrices were found in the inducedgroups than the control groups. (3) HE staining showed some roundcells in pores of scaffolds, but the cell density inner was lower thanouter. The extracellular matrix was stained positively for Safranin O/Fast Green staining, Masson staining after initial chondrogenic inductionin the induced groups, and indicated collagenâ…¡and aggrecan wereexpressed positively, still a few cartilage lacunae to be found. Butnegatively in the contron groups. (4) RT-PCR indicated that theinduced groups could express Col2al mRNA, aggrecan mRNA and Sox9mRNA at 3 weeks after initial chondrogenic induction, but no express incontrol groups.Conclusion Adipose-derived mesenchymal stem cells may becultured with PLGA scaffolds, still form a material with part character ofcartilaginous tissue and express cartilage phenotype under the specificinducing environment in vitro. Chapterâ…£Heterotopic chondrogenesis of adipose-derivedmesenchymal stem cells in PLGA scaffolds in vivoObjective To study Heterotopic chondrogenesis of adipose-derived mesenchymal stem cells in PLGA scaffolds in vivo, which hadexpressed cartilage phenotype under the specific inducing environment invitro.Methods (1) Adipose -derived mesenchymal stem cells loadingin PLGA were induced or noninduced for three weeks according to theChapterâ…¢. (2) The induced complexes of cells and PLGA scaffolds andthe noninduced ones were implanted into the zygomorphic backsubcutaneous layer of nude mice respectively, still were studied in theface of texture and shape changes. The subsistence of nude mice wasevaluated. (3) The complexes of cells and scoffolds was terminated invivo, embeded in paraffin and cut in section, still assessed using H-Estaining, Alcian blue staining, Toluidine blue staining, Safranin O/ FastGreen staining and Masson staining, still collagenâ…¡immunohistochemistry and aggrecan immunofluorescence at 8, 12weeks after they were impanted in vivo. (4) The mRNA expressions ofCol2al, Agc1 and Sox9 were detected by RT-PCR at 8, 12 weeks afterthe complex were impanted in vivo.Results (1) The subsistence of nude mice was well, nopostoperative toxic and inflammatory reaction. The induced groups maysustain their original size, the tenacity increased. But the noninducedgroups disappeared progressively. (2) The extracellular matrix of theinduced groups was stained positively for Alcian blue, Toluidine blue,Safranin O/ Fast Green and Masson at 8,12 weeks postoperatively, andindicated collagenâ…¡and aggrecan were expressed positively. Manycartilage lacunae were found. (3) RT-PCR indicated that the inducedgroups implanted in vivo could express Col2al mRNA, aggrecanmRNA and Sox9 mRNA at 8, 12 weeks postoperatively.Conclusion Adipose-derived mesenchymal stem cells combinedwith PLGA scaffolds induced in vitro have the capability of Heterotopic chondrogenesis in vivo.
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