Chondrogenesis in Three-Dimensional Culture of? ?Bone Marrow Derived ?Murine Mesenchymal Stem Cells within an Alginate Gel
Trauma Monthly: 12 (2); 97-106 Article Type: Research Article
January 1, 2007
January 1, 2007
L. Chondrogenesis in Three-Dimensional Culture of? ?Bone Marrow Derived ?Murine Mesenchymal Stem Cells within an Alginate Gel,
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Objective. Study of the chondrogenesis of the marrow derived murine mesenchymal stem cells (MSCs) embedded three-dimensionally within an alginate gel. Material and Methods. 4-6 week old NMRI mice were killed, the bone marrow cells were harvested, counted and 500 cells of which were plated per each well in 6-well-cell-culture plate. Fibroblastic cells were then purified and expanded through two successive subcultures. In order to prepare three-dimensional culture systems , 10 million of passaged-2-fibroblastic cells were suspended in 10 ml of alginate solution and the cell suspension were divided into 10 equal part each of which were placed per well of 12-well-culture plate. MSCs-alginate suspension were exposed to CaCl solution by which the gelation process was occurred .After that the CaCl was removed and the cultures were provided by a chondrogenic differentiation medium containing 10ng TGF-B3 and 10 ng BMP-6 for 3 weeks after which the occurrence of the differentiation were evaluated by toloidin blue staining , RT-PCR analysis and transmission electron microscopy. In present study the mesenchymal nature of the isolated cells were evaluated by differentiation into bone and adipocytic cell lineage. Results. In primary culture, variety of cell morphology ranging from the polygonal cells to the elongated, flattened and spindly–shaped cells was observed. The pure fibroblastic cells were not appeared until time the first subculture was performed. Enough number of the fibroblastic cells was then available for conducting the remaining part of experiment after the second round of cell passage was done. The result of the differentiation evaluation by toloidine blue staining method indicated that the MSCs in alginate assumed a rounded morphology with a lacunae around them.The cells have been surrounded by a methachromatic matrix .RT-PCR analysis revealed that the mRNA of collagen II, X and agreacan have been largely produced in differentiated cells. Ultra thin sections showed the cells with well-developed secretive organelles indicative of their active chondroblastic state rather than non-active chondrocytic state. Conclusion. MSCs could acquire cartilage-cell phenotype when being cultivated within the alginate gel in a three-dimensional culture system in the absence of cell condensation, an important prerequisite for chondrogenesis during development.
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