Abstract: Objective To explore the methods of culture and induction of bone marrow stem cells（BMSCs） in vitro and fabrication of cell sheet of BMSCs. Methods Bone marrow stem cells were isolated with the method of density gradient centrifugation from canine and cultured. BMSCs were induced to differentiate to osteoblasts. The BMSCs were observed under inverted microscope and scanning electron microscope. The BMSCs after induced were transferred to the temperature-responsive culture dishes and cultured in incubator at 37 ℃, 5% CO2 and saturated humidity. The temperature of the incubator was changed from 37 ℃ to 20 ℃, to fabricate the cell sheet of BMSCs. Results BMSCs was fusiform and grew in whirl-like arrangement while the BMSCs induced were oval or polygonal; when the temperature dropped to 20 ℃, bone marrow stem cell sheet detached automatically from the temperature-responsive culture dishes. Conclusion BMSCs can be effectively obtained with the method of density
gradient centrifugation and induced to differentiate into osteoblasts. Bone marrow stem cell sheet layer can be used to wrap the scaffold for construction of functional osteogenesis.

Key words: bone marrow stem cells, cell sheet, temperature-responsive culture dishes