To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified ...To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.展开更多
文摘To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.
