| ObjectivesFreuentley,Low back pain is mainly caused by the degenerationg of the intervertebral disc.Disc degeneration begins with a loss of disc cells and alterations in the extracellular matrix of the disc.Although the lineage of the different cell types in the intervertebral disc is not known with certainty,it is ageed that ch- ondrocytes and cells of the nucleus pulposus share many common features.Recently,One promising approach for this problem involves the use of cells transplanted to the degenerative disc to achieve functional tissue repair Because MSC can differentiate into chondrocytelike cells that are phenotypically similar to the nucl- eus pulposus.The majority of scholars major in studying bone marrow-derived cells which can be differentiated into NP-like cells using microenvironmental con ditions.Recently,In addition to bone marrow derived cells,Adipose tissue represents an abundant and accessible source of adipose stem cells with the ability to differentiate into chondrocytelike cells along multiple pathways,for instance,transgenic techniques,techniques of differentiating adipose stem cells into chondrocytelike cells combining different grow factors,so we have designed this study to explore the changes of rabbit Adipos Stem Cells(ASCs) and Bone Mesenchymal Stem Cells(BMSCs) cultured in vitro and the anabolic effect introduced by Transforming Growth Factor-β1 (TGF-β1) and basic Fibroblast Growth Factor(BFGF) on the cells.Methods1.BMSC s were cultured and Adipose tissue,excised from the suprascapular fat pad Of White rabbits,was minced in a 0.075%collaenase typeⅡsolution for 1 hour at 37℃.The digested tissue was filtered and centrifuged to collect a pellet of ASCs.BMSCs and ASCs are cultrred with DMEM,DMEM/F12(2:1),α—MEM respectively.the Morphological changes of BMSCs and ASCs were observed by inverted microscope daily,and cell number were analyzed by comparing between ASCs and BMSC s.2.After expansion to passage 3,the ASCs were collected via trypsinization and seeded onto 24cm2 culture flask at a density of 2×105/ml,which were induced in CM which is consisting of DMEM/F12(2:1) supplemented with 1.25mg/ml bovine serum albumin(BSA),10ng/ml transforming growth factor-β1(TGF-β1;Gibco),100 nmol/L dexamethasone,50μg/mL ascorbate,100μg/mL sodium pyruvate,40μg/mL proline,and 1%ITS-PLUS(10 mg/ml insulin,6.7 mg/ml sodium selenite,5.5 mg/ml transferrin and 2 ng/ml ethanolamine) DMEM/F12(2:1)3.Rabbit BMSCs and ASCs grouped into 4 goups were cultured in induced media for three weeks:group A:BMSCs-CM;group B:BMSCs-CM-5ng/mlbFGF;group C:ASCs-CM;group D:ASCs+CM-5ng/mlbFGF. Total aggecan synthesis was assayed by 35SO42- incorporation,Total collagen synthesis was assayed by measuring total hydroxyproline.Results1.BMSCs and ASCs show much higher gowth rate when cultured inα—MEM medium,in comparison with DMEM,DMEM/F12(2:1).2.In monolayer:two stem cells attachhment cutured in monolayer is greatly enhanced and cell clones are abtmdant while the cells attachment is rather difficult and cell clones are less when cutured in CM.3.Two stem cells possess a round-like morphology when cultured in all goups while the quantity of cells cutured in the group B and goup D is more than that of cells cutured in the other two groups.Total aggrecan and total hydroxyproline synthesis of goup B is increased as compared with goup A,and Total aggrecan and total hydroxyproline synthesis of group D is increased as compared with goup C.while Total aggrecan and total hydroxyproline synthesis of group D have no diference,as compared with the which of group B.Conclusionin the cultivation of Adipos Stem Cells and Bone Mesenchymal Stem Cells,α—MEM medium was superior to DMEM and DMEM/F12(2:1).Cultured in CM suppling with BFGF conditions,the rabbit Adipos Stem Cells and Bone Mesenchymal Stem Cells grow well and metabolism is enhanced Adipos Stem Cells may be substitute of Bone Mesenchymal Stem Cells for nucleus pulposus tissue engineering.
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